Compositions and methods for transgenic crops resistant to thaxtomins

ABSTRACT

The present disclosure relates to compositions comprising nitroreductase enzymes; plants, plant calluses, plant seeds, and vegetables incorporating genes expressing nitroreductase enzymes; methods for stably and operably incorporating genes expressing nitroreductase enzymes into plants and plant tissues; wherein the nitroreductase enzymes are capable of reducing nitro groups on phytotoxic and/or otherwise harmful compounds such as, for example, thaxtomins, wherein the thaxtomins are secreted by plant pathogenic bacteria and/or exogenously applied as an agricultural composition such as an herbicide, and wherein reducing the nitro groups renders the thaxtomins non-damaging or reduces the level of damage from the thaxtomins to the plants expressing the nitroreductase enzymes.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/946,069, filed Dec. 10, 2019, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically as a text file and is hereby incorporated byreference in its entirety. Said text file, created on Dec. 8, 2020, isnamed 222107-2630_ST25.txt and is 5,093 bytes in size.

FIELD

The present disclosure relates to compositions and methods fortransgenic crops resistant to thaxtomins.

BACKGROUND

Pathogens and pests are estimated to lead to 17.2%-30% yield losses ofvarious crops globally. Accordingly, synthetic pesticides, fungicides,and antimicrobials have been developed to fight against pathogens andpests. These chemicals have achieved tremendous successes but have alsocaused lasting issues, e.g., emerging resistance, environment pollution,and the killing of beneficial insects. In the past decades, several newstrategies have emerged to address the above-mentioned issues. Forexample, chemicals that are originated from natural products haveincreasingly been developed for crop protection, as they can offer newmechanisms of action, can act on new targets, and have minimal to noenvironmental impacts. Genetic engineering of crops to introduce newtraits for crop protection has become another successful strategy inrecent years. For example, Roundup Ready® crops are resistant toRoundup® (glyphosate), which is the most widely used herbicide.

Streptomyces scabies and several other Streptomyces species are grampositive, filamentous bacterial plant pathogens that induce diseases tobroadleaf crops (e.g., potato, radish, and onion). Among these diseases,potato common scab is the most severe and widespread and incurs enormouseconomic losses to farmers. Almost all known plant pathogenicStreptomyces strains produce thaxtomins. Purified thaxtomins alone areable to induce common scab disease symptoms and are known virulentfactors of plant pathogens. Importantly, thaxtomins demonstrate a novelmechanism of action, inhibiting cellulose biosynthesis at the nM rangeand have been developed as bioherbicides for weed control. Variousmethods have been explored to cure potato scab disease caused bythaxtomin-producing pathogenic Streptomyces strains. A somatic cellselection approach has been developed using thaxtomin A as a positiveselection agent to obtain Solanum tuberosum variants with significantlyimproved resistance to common scab disease but has met with limitedsuccess. Furthermore, the synthetic compound 2,4-dichlophenoxyaceticacid has been used to fight the disease but its adverse effects to humanhealth make it problematic. In recent years, many biocontrol strategieshave been developed to control common scab of potato by using microbialspecies including Pseudomonas sp. LBUM 22322-24, Bacillus altitudinisstrain AMCC 10130425, Bacillus amyloliquefaciens BAC0326-29,StreptomycesA1RT30, and Streptomyces violaceusnigerAC12AB31. However,these biocontrol strategies require extra efforts to maintain strainbalance. Therefore, there is a lasting need for new agents andstrategies for controlling potato common scab.

Several studies have demonstrated that the nitro group of thaxtomins isessential to their virulent and herbicidal activities. Thetransformation of the nitro group of thaxtomins thus stands out as apotential promising strategy for controlling potato common disease.Although chemical reduction of the nitro group of thaxtomins into aminegroup is possible, the chemical reaction is not compatible with livingorganisms such as crops. In this regard, biological approaches,specifically enzymatic reduction of the nitro group, are moreenvironmentally sound. More importantly, genetic engineering of cropswith the corresponding genes has a promise to confer resistance tothaxtomins as well as thaxtomin-producing plant pathogens in theresulting crops.

Flavin mononucleotide-dependent nitroreductases form an enzymesuperfamily containing more than 24,000 sequences from all domains oflife. These enzymes catalyze a diverse range of reactions and some mayinitiate the catabolism of nitroaromatic compounds. For example, severalprevious studies have shown that the nitroreductase NfsB from differentmicrobial species can reduce the nitro group of many synthetic chemicalsand natural products. Very recently, the nitroreductase NfsB fromHaemophilus influenzae was shown to catalyze the reduction of the nitrogroup of a series of nitro-containing chemicals. Furthermore, previousstudies have shown that some bacterial and fungal species may expressnitroreductases to degrade thaxtomins, although specific enzymes havenot been identified. All of these studies indicated that nitroreductasescan provide a way to reduce the nitro group of thaxtomins, therebydisarming thaxtomins' virulent and herbicidal activity.

Despite advances in research relating nitroreductase proteins, there isstill a scarcity of strategies for combating diseases ofcommercially-important crops caused by thaxtomins. Furthermore, althoughthaxtomins are effective, naturally-occurring herbicides, they cannotcurrently be used as such due to off-target damage to crop plants. Itwould be desirable to produce thaxtomin-resistant plants in order toenable use of thaxtomins as herbicides. These needs and other needs aresatisfied by the present disclosure.

SUMMARY

In accordance with the purpose(s) of the present disclosure, as embodiedand broadly described herein, the present disclosure, in one aspect,relates to compositions comprising nitroreductase enzymes; plants, plantcalluses, plant seeds, and vegetables incorporating genes expressingnitroreductase enzymes; methods for stably and operably incorporatinggenes expressing nitroreductase enzymes into plants and plant tissues;wherein the nitroreductase enzymes are capable of reducing nitro groupson phytotoxic and/or otherwise harmful compounds such as, for example,thaxtomins, wherein the thaxtomins are secreted by plant pathogenicbacteria and/or exogenously applied as an agricultural composition suchas an herbicide, and wherein reducing the nitro groups renders thethaxtomins non-damaging or reduces the level of damage from thethaxtomins to the plants expressing the nitroreductase enzymes.

Other systems, methods, features, and advantages of the presentdisclosure will be or become apparent to one with skill in the art uponexamination of the following drawings and detailed description. It isintended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present disclosure, and be protected by the accompanying claims. Inaddition, all optional and preferred features and modifications of thedescribed embodiments are usable in all aspects of the presentdisclosure taught herein. Furthermore, the individual features of thedependent claims, as well as all optional and preferred features andmodifications of the described embodiments are combinable andinterchangeable with one another.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the present disclosure can be better understood withreference to the following drawings. The components in the drawings arenot necessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIG. 1 shows enzymatic transformation of thaxtomins into 4-aminothaxtomins by the nitroreductase NfsB.

FIG. 2 shows SDS-PAGE analysis of purified recombinant NfsB. M: marker.

FIGS. 3A-3B show HPLC and LC-MS spectra of 4-amino-thaxtomins producedin the NfsB reactions. FIG. 3A. HPLC spectra of the transformations ofthaxtomins A to D. The chemicals were detected at both 280 nm and 380 nm(specific to the nitro group). FIG. 3B. LC-MS detection of the [M+1]⁺peak of compound 5, which is indicated in the box. FIG. 3C. LC-MSdetection of the [M+1]⁺ peak of compound 6, which is indicated in thebox. FIG. 3D. LC-MS detection of the [M+1]⁺ peak of compound 7, which isindicated in the box. FIG. 3E. LC-MS detection of the [M+1]⁺ peak ofcompound 8, which is indicated in the box.

FIGS. 4A-4E show NMR spectra of 4-amino thaxtomin A. FIG. 4A: ¹H NMR(600 MHz; CD₃OD); FIG. 4B: ¹³C NMR (150 MHz; CD₃OD); FIG. 4C: COSY; FIG.4D: HSQC; FIG. 4E: HMBC.

FIG. 5 shows radish seedling assay results. 1: DMSO; 2: 2.0 μM4-amino-thaxtomin A; 3: 0.05 μM 4-amino-thaxtomin A; 4: 2.0 μM thaxtominA; 5: 0.05 μM thaxtomin A.

FIG. 6 shows a modeled structure of NfsB from Haemophilus influenzaprepared using SWISS-MODEL with an NADPH-dependent enzyme from Vibriofischeri as the template (PDB ID: 1VFR). The structures of NfsB (labeledas “C”) and the template (labeled as “D”) were overlaid; the two loopslabeled as “A” and “B” play key roles in catalysis and substratebinding.

FIG. 7A shows SDS-PAGE analysis of purified recombinant wild type (WT)and NsfB R20A mutant. Both proteins showed the expected molecular weightat around 26 kDa. FIG. 7B shows the relative catalytic activity of wildtype (WT) and NsfB R20A mutant in converting thaxtomin A under the samereaction conditions. The reaction product was analyzed by LC-MSanalysis. The peak areas of amino-thaxtomine A produced in the WT-fullreaction was set as 100% to normalize the relative activities of themutant enzyme. The data represent the mean±standard deviation of atleast two independent experiments.

Additional advantages of the present disclosure will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or can be learned by practice of the presentdisclosure. The advantages of the present disclosure will be realizedand attained by means of the elements and combinations particularlypointed out in the appended claims. It is to be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of the presentdisclosure, as claimed.

DETAILED DESCRIPTION

Many modifications and other embodiments disclosed herein will come tomind to one skilled in the art to which the disclosed compositions andmethods pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the present disclosures are not to be limited to thespecific embodiments disclosed and that modifications and otherembodiments are intended to be included within the scope of the appendedclaims. The skilled artisan will recognize many variants and adaptationsof the aspects described herein. These variants and adaptations areintended to be included in the teachings of this disclosure and to beencompassed by the claims herein.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure.

Any recited method can be carried out in the order of events recited orin any other order that is logically possible. That is, unless otherwiseexpressly stated, it is in no way intended that any method or aspect setforth herein be construed as requiring that its steps be performed in aspecific order. Accordingly, where a method claim does not specificallystate in the claims or descriptions that the steps are to be limited toa specific order, it is no way intended that an order be inferred, inany respect. This holds for any possible non-express basis forinterpretation, including matters of logic with respect to arrangementof steps or operational flow, plain meaning derived from grammaticalorganization or punctuation, or the number or type of aspects describedin the specification.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present disclosure is not entitled to antedate such publicationby virtue of prior disclosure. Further, the dates of publicationprovided herein can be different from the actual publication dates,which can require independent confirmation.

While aspects of the present disclosure can be described and claimed ina particular statutory class, such as the system statutory class, thisis for convenience only and one of skill in the art will understand thateach aspect of the present disclosure can be described and claimed inany statutory class.

It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosed compositions andmethods belong. It will be further understood that terms, such as thosedefined in commonly used dictionaries, should be interpreted as having ameaning that is consistent with their meaning in the context of thespecification and relevant art and should not be interpreted in anidealized or overly formal sense unless expressly defined herein.

Prior to describing the various aspects of the present disclosure, thefollowing definitions are provided and should be used unless otherwiseindicated. Additional terms may be defined elsewhere in the presentdisclosure.

Definitions

As used herein, “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps, or components, or groups thereof.Moreover, each of the terms “by”, “comprising,” “comprises”, “comprisedof,” “including,” “includes,” “included,” “involving,” “involves,”“involved,” and “such as” are used in their open, non-limiting sense andmay be used interchangeably. Further, the term “comprising” is intendedto include examples and aspects encompassed by the terms “consistingessentially of” and “consisting of.” Similarly, the term “consistingessentially of” is intended to include examples encompassed by the term“consisting of.”

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a thaxtomin,” “acrop plant,” or “a nitroreductase enzyme,” including, but not limitedto, combinations of two or more such thaxtomins, crop plants, ornitroreductase enzymes, and the like.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. Ranges can be expressed herein as from “about” one particularvalue, and/or to “about” another particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms a furtheraspect. For example, if the value “about 10” is disclosed, then “10” isalso disclosed.

When a range is expressed, a further aspect includes from the oneparticular value and/or to the other particular value. For example,where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe present disclosure, e.g. the phrase “x to y” includes the range from‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. Therange can also be expressed as an upper limit, e.g. ‘about x, y, z, orless’ and should be interpreted to include the specific ranges of ‘aboutx’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’,less than y′, and ‘less than z’. Likewise, the phrase ‘about x, y, z, orgreater’ should be interpreted to include the specific ranges of ‘aboutx’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’,greater than y′, and ‘greater than z’. In addition, the phrase “about‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’to about ‘y’”.

It is to be understood that such a range format is used for convenienceand brevity, and thus, should be interpreted in a flexible manner toinclude not only the numerical values explicitly recited as the limitsof the range, but also to include all the individual numerical values orsub-ranges encompassed within that range as if each numerical value andsub-range is explicitly recited. To illustrate, a numerical range of“about 0.1% to 5%” should be interpreted to include not only theexplicitly recited values of about 0.1% to about 5%, but also includeindividual values (e.g., about 1%, about 2%, about 3%, and about 4%) andthe sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%;about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and otherpossible sub-ranges) within the indicated range.

As used herein, the terms “about,” “approximate,” “at or about,” and“substantially” mean that the amount or value in question can be theexact value or a value that provides equivalent results or effects asrecited in the claims or taught herein. That is, it is understood thatamounts, sizes, formulations, parameters, and other quantities andcharacteristics are not and need not be exact, but may be approximateand/or larger or smaller, as desired, reflecting tolerances, conversionfactors, rounding off, measurement error and the like, and other factorsknown to those of skill in the art such that equivalent results oreffects are obtained. In some circumstances, the value that providesequivalent results or effects cannot be reasonably determined. In suchcases, it is generally understood, as used herein, that “about” and “ator about” mean the nominal value indicated ±10% variation unlessotherwise indicated or inferred. In general, an amount, size,formulation, parameter or other quantity or characteristic is “about,”“approximate,” or “at or about” whether or not expressly stated to besuch. It is understood that where “about,” “approximate,” or “at orabout” is used before a quantitative value, the parameter also includesthe specific quantitative value itself, unless specifically statedotherwise.

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired modification of a physical property ofthe composition or material. For example, an “effective amount” of asherbicidal composition refers to an amount that is sufficient to achievethe desired control of target weeds. The specific level in terms ofgrams per acre in a composition required as an effective amount willdepend upon a variety of factors including the amount and type of levelof weed growth, stage of weed growth, and stage of crop plant growth.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, “phytotoxic” refers to a deleterious effect of acompound on plant growth. In one aspect, application of a phytotoxiccompound can kill all or a portion of a plant, can slow or stunt aplant's growth, can damage a plant organ such as a leaf, stem, root,fruit, flower, or the like, can inhibit seedling growth, or acombination thereof.

“Transgenic” as used herein refers to an organism that contains genes orgenetic material from an unrelated organism (“exogenous DNA”). In afurther aspect, the exogenous DNA in a transgenic organism can beartificially introduced in a laboratory setting. In a still furtheraspect, the exogenous DNA in a transgenic organism can be introducedinto the organism's cells as part of a plasmid, or can be introducedinto the organism's genome or the genome of a plastid (e.g.,chloroplast) or mitochondrion using any technique known in the artincluding, but not limited to use of a biolistic device or a genomeediting technique such as, for example, use of an engineered nuclease(i.e., meganucleases, zinc finger nucleases, transcriptionactivator-like effector-based nucleases, and/or CRISPR/Cas9). In oneaspect, exogenous DNA is introduced into plant callus from which plantsincorporating the exogenous DNA can later be grown.

A “recombinant organism” is an organism containing anartificially-induced mutation distinguishing it from wild-typeorganisms. In one aspect, a recombinant organism can incorporate a genefrom an unrelated species that has been introduced by geneticmodification or genome editing techniques, or can incorporate a plasmidthat has been so engineered. In a further aspect, disclosed herein arerecombinant plants incorporating nitroreductase enzymes.

As used herein, a “primer” is a short nucleic acid sequence thatprovides a starting point for DNA synthesis (for example, for use in thepolymerase chain reaction). Primers useful herein can be synthesized byany technique known in the art including solid-phase synthesis usingphosphoramidite chemistry. In one aspect, SEQ ID NO: 2 and SEQ ID NO: 3represent primers useful for scaling-up synthesis of one nitroreductasegene useful in the processes disclosed herein. Ideal primers havesequence overlap with the DNA that is desired to be inserted into thecell but do not form secondary or tertiary structures (i.e., hairpins,G-quadruplexes, or the like).

An “expression vector” is typically a plasmid or virus designed tointroduce one or more target genes into a cell. In one aspect, anexpression vector contains regulatory sequences (i.e., enhancers,promoters, and the like) that interact with the host cell's proteinsynthesis machinery to produce the proteins encoded by the target genes.

As used herein, a “plasmid” is a DNA molecule within a cell that is notpart of the cell's chromosomal DNA. Plasmids are typically circular anddouble-stranded and are most commonly found in bacteria but can, in somecases, be present in archaea or eukaryotes (i.e., plasmids in yeasts orTi-plasmids for introducing genes into plants).

“Transformation” as used herein is genetic alteration of a cell byuptake of DNA from its surroundings. In some aspects, transformation isaccomplished artificially. In one aspect, transformed cells can expressproteins encoded by the exogenous DNA. In some aspects, transformationas it relates to eukaryotic cells (such as, for example, plants) may bereferred to as “transfection.”

A “restriction enzyme” (also known as a restriction endonuclease) is anenzyme that cleaves double-stranded DNA at a specific recognition site.Restriction enzymes are native to bacteria and archaea and many can bepurchased commercially for use in genetic engineering applications.Restriction enzymes may cut such that there are overhanging or “sticky”ends or such that there are “blunt” ends with no overhang. In oneaspect, use of restriction enzymes allows cleavage of a plasmid or otherexpression vector for the purpose of inserting a gene of interest.

A “recognition site” is a double-stranded length of DNA, typically 4 to8 base pairs long, that a restriction enzyme must recognize in order tocleave DNA. A “restriction site” is the location at which the DNA iscleaved. A restriction site and a recognition site may be the same(i.e., the enzyme cleaves the DNA at the recognition site) or different(i.e., the enzyme cleaves the DNA some distance away from therecognition site). Recognition sites are typically palindromic (eithermirror-like palindromes or inverted-repeat palindromes). In one aspect,plasmids typically include restriction sites to aid in the insertion ofexogenous genes into the plasmids.

The term “agriculturally acceptable” is used herein to includeagricultural, industrial and residential uses which are compatible withplants.

As used herein, the terms “controlling” and “combating” are synonyms. Asused herein, by “controlling a pest” or “controls a pest” is intendedany effect on a pest that results in limiting the damage that the pestcauses. Controlling a pest includes, but is not limited to, killing thepest, inhibiting development of the pest, altering fertility or growthof the pest in such a manner that the pest provides less damage to theplant, or in a manner for decreasing the number of offspring produced,producing less fit pests, producing pests more susceptible to predatorattack, or deterring the pests from colonizing the plant. In one aspect,the pest is a microorganism such as, for example, a pathogenicmicroorganism and/or a microorganism that secretes one or morephytotoxic compounds.

As used herein, the terms “undesirable vegetation”, “harmful plants” and“weeds” are synonyms.

As used herein, an “herbicide” is a phytotoxic compound deliberatelyapplied to an area to destroy undesired vegetation (e.g., “weeds”). Inone aspect, herbicides are useful for application to agricultural fieldsso that weeds do not compete with crop plants for resources. In anotheraspect, a compound is useful as an herbicide if it causes damage toundesired vegetation but not to the agricultural crop or other desiredvegetation.

As used herein, the term “herbicide resistant” refers to plants that areresistant to herbicides for example, but not limited to, glyphosate,dicamba, 2,4-dichlorophenoxyethanoic acid, glufosinate, ACCaseinhibitors, HPPD inhibitors, acetohydroxyacid synthase inhibitors,thaxtomins, and combinations thereof.

“Adjuvants” are materials that facilitate the activity of herbicides orthat facilitate or modify characteristics of herbicide formulations orspray solutions.

As used throughout this application, the term “agriculturally acceptablesalt” refers to a salt comprising a cation that is known and accepted inthe art for the formation of salts for agricultural or horticulturaluse. In one aspect, the salt is a water-soluble salt.

The “crops of useful plants” to be protected typically comprise, forexample, the following species of plants: cereals (wheat, barley, rye,oats, maize (including field corn, popcorn and sweet corn), rice,sorghum and related crops); beet (sugar beet and fodder beet);leguminous plants (beans, lentils, peas, soybeans); oil plants (rape,mustard, sunflowers); cucumber plants (marrows, cucumbers, melons);fiber plants (cotton, flax, hemp, jute); vegetables (spinach, lettuce,asparagus, cabbages, carrots, eggplants, onions, pepper, tomatoes,potatoes, paprika, okra); plantation crops (bananas, fruit trees, rubbertrees, tree nurseries), ornamentals (flowers, shrubs, broad-leaved treesand evergreens, such as conifers); as well as other plants such asvines, bushberries (such as blueberries), caneberries, cranberries,peppermint, rhubarb, spearmint, sugar cane and turf grasses including,for example, cool-season turf grasses (for example, bluegrasses (PoaL.), such as Kentucky bluegrass (Poa pratensis L.), rough bluegrass (Poatrivialis L.), Canada bluegrass (Poa compressa L.) and annual bluegrass(Poa annus L.); bentgrasses (Agrostis L.), such as creeping bentgrass(Agrostis palustris Huds.), colonial bentgrass (Agrostis tenius Sibth.),velvet bentgrass (Agrostis canina L.) and redtop (Agrostis alba L.);fescues (Festuca L.), such as tall fescue (Festuca arundinacea Schreb.),meadow fescue (Festuca elatior L.) and fine fescues such as creeping redfescue (Festuca rubra L.), chewings fescue (Festuca rubra var. commutateGaud.), sheep fescue (Festuca ovina L.) and hard fescue (Festucalongifolia); and ryegrasses (Lolium L.), such as perennial ryegrass(Lolium perenne L.) and annual (Italian) ryegrass (Lolium multiflorumLam.)) and warm-season turf grasses (for example, Bermudagrasses(Cynodon L. C. Rich), including hybrid and common Bermudagrass;Zoysiagrasses (Zoysia Willd.), St. Augustinegrass (Stenotaphrumsecundatum (Walt.) Kuntze); and centipedegrass (Eremochloa ophiuroides(Munro.) Hack)).

The term “useful plants” also includes useful plants that have beenrendered tolerant to herbicides like bromoxynil or classes of herbicides(such as, for example, HPPD inhibitors, ALS inhibitors, for exampleprimisulfuron, prosulfuron and trifloxysulfuron, EPSPS(5-enol-pyrovyl-shikimate-3-phosphate-synthase) inhibitors, GS(glutamine synthetase) inhibitors, thaxtomins, and/or PPO(protoporphyrinogen-oxidase) inhibitors) as a result of conventionalmethods of breeding or genetic engineering. An example of a crop thathas been rendered tolerant to imidazolinones, e.g. imazamox, byconventional methods of breeding (mutagenesis) is Clearfield® summerrape (Canola). Examples of crops that have been rendered tolerant toherbicides or classes of herbicides by genetic engineering methodsinclude glyphosate- and glufosinate-resistant maize varietiescommercially available under the trade names RoundupReady®, Herculex I®and LibertyLink®.

The term “useful plants” also includes useful plants which have been sotransformed by the use of recombinant DNA techniques that they arecapable of synthesizing one or more selectively acting toxins, such asare known, for example, from toxin-producing bacteria, especially thoseof the genus Bacillus.

The term “useful plants” also includes useful plants which have been sotransformed by the use of recombinant DNA techniques that they arecapable of synthesizing antipathogenic substances having a selectiveaction, such as, for example, the so-called “pathogenesis-relatedproteins” (PRPs, see e.g. EP-A-0 392 225). Examples of suchantipathogenic substances and transgenic plants capable of synthesizingsuch antipathogenic substances are known, for example, from EP-A-0 392225, WO 95/33818, and EP-A-0 353 191. The methods of producing suchtransgenic plants are generally known to the person skilled in the artand are described, for example, in the publications mentioned above.

Unless otherwise specified, temperatures referred to herein are based onatmospheric pressure (i.e. one atmosphere).

Thaxtomins

2,5-Diketopiperazines (DKPs) are a family of small cyclopeptides made oftwo amino acid monomers. The DKP scaffold is a privileged structure fordrug discovery and other applications as it is metabolically stable,structurally constrained, and amenable to multiple stereo-specificmodifications. “Thaxtomins” are phytotoxic secondary2,5-diketopiperazine metabolites produced in plant pathogenicStreptomyces strains and have received considerable interests asbioherbicides due to their ability to inhibit cellulose biosynthesis inthe nanomolar range (see FIG. 1 ).

Thaxtomins include a 4-nitroindole moiety that renders them phytotoxic.In one aspect, thaxtomins are produced by pathogenic Streptomycesspecies including, but not limited to, S. scabies, S. turgidiscabies, S.acidiscabies, S. luridiscabiei, S. puniciscabiei, S. nieviscabei, S.ipomoea, and other related species. In a further aspect, thaxtominscause necrosis in plants by inhibiting cellulose synthase. In oneaspect, thaxtomins are virulence factors in the disease known as commonscab of potato and may also affect sweet potato, beet, carrot, parsnip,radish, rutabaga, turnip, and other commercially-important root crops.In another aspect, thaxtomins can inhibit growth of monocot and dicotseedlings. In still another aspect, thaxtomins are useful as pre- andpost-emergent herbicides for broadleaf weeds, sedges, and grassy weeds.

Nitroreductase Enzymes

“Nitroreductases” are members of a family of enzymes that reducenitrogen-containing compounds, especially compounds having a nitrofunctional group. Many nitroreductase enzymes require cofactorsincluding, but not limited to, one or more of flavin mononucleotide(FMN), flavin adenine dinucleotide (FAD), nicotinamide adeninedinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP),and the like. In one aspect, a nitroreductase enzyme can reduce a nitrogroup to an amino group via two putative intermediates. In a furtheraspect, this reduction can cause a phytotoxic compound such as, forexample, a thaxtomin, to lose its phytotoxicity.

In one aspect, disclosed herein is a nitroreductase with specificity forthaxtomin A. In a further aspect, thaxtomin A is the chief thaxtominspecies secreted by S. scabies, and is primarily responsible for damagecaused by potato common scab. In a still further aspect, thenitroreductase can deactivate thaxtomin A by reducing the nitro group ofthaxtomin A to an amino group. In one aspect, the nitroreductase gene isisolated from Haemophilus influenzae and has SEQ ID NO: 1 or is aderivative or variant thereof such as, for example, a cDNA copyconsisting of nitroreductase exons stitched together after introns havebeen removed. In a further aspect, the nitroreductase gene produces aprotein having SEQ ID NO: 4 or a derivative or variant thereof. Inanother aspect, the nitroreductase protein is known as NfsB. In oneaspect, the residues involved in substrate binding may include at leastone of R20, W71, S46, C164, G72, G168, E167, K119, Q76, P165, S46, andK106. In another aspect, the residues involved in interacting with FMNinclude at least one of R16, R209, K207, S18, S45, and R20. In oneaspect, DNA and protein sequences useful herein are provided in Table 1:

TABLE 1 Genes and Sequences Useful Herein SEQ  ID  Description SequenceNO: NfsB gene ATGACTCAACTTACTCGTGAACA 1 (HaemophilusAGTTCTTGAACTCTTCCATCAAC influenzae) GCAGCTCAACACGTTATTACGACCCAACAAAAAAAATCAGTGATGA AGATTTTGAATGTATTTTAGAGT GCGGTCGATTATCGCCGAGTTCTGTAGGCTCTGAGCCTTGGAAATT TTTAGTGATTCAAAATAAAACCT TACGCGAAAAAATGAAACCTTTTAGCTGGGGAATGATAAATCAGCT TGATAATTGCAGTCATCTTGTGG TAATTCTCGCGAAGAAAAATGCCCGTTATGATAGTCCGTTTTTTGT GGATGTGATGGCACGCAAAGGCT TGAACGCAGAGCAACAACAAGCCGCCCTCACAAAATACAAAGCCCT GCAAGAAGAAGATATGAAATTAC TCGAAAACGACCGCACTTTATTTGATTGGTGCAGCAAACAAACTTA TATCGCCCTTGCAAATATGCTTA CTGGAGCTTCAGCCCTTGGCATCGACTCTTGCCCAATTGAAGGTTT TCATTACGACAAAATGAATGAAT GCCTCGCCGAAGAAGGATTATTCGATCCTCAAGAATATGCGGTTTC TGTCGCCGCAACCTTTGGCTATC GCTCACGCGATATTGCGAAAAAATCCCGTAAAGGATTGGATGAAGT GGTGAAATGGGTGGGGTAA NfB-NdeI-F ACTCATATGACTCAACTTACTCG 2 primer TGAA NfB-HindIII-ACTAAGCTTCCCCACCCATTTCA 3 R primer CCACTTCA NfsB MTQLTREQVLELFHQRSSTRYYD 4 protein PTKKISDEDFECILECGRLSPSSVGSEPWKFLVIQNKTLREKMKPF SWGMINQLDNCSHLVVILA

FDWCSKQTYIALANMLTGASALG IDSCPIEGFHYDKMNECLAEEGL FDPQEYAVSVAATFGYRSRDIAKKSRKGLDEVVKWVG NfsB  KTLREKMKPFSWGMINQLDN 5 protein  putative catalytic domain A NfsB  KKNARYDSPFFVDVMARKGLNAE 6 protein QQQAALTKYKALQEEDMKLLEND putative RTL catalytic  domain B

In one aspect, the underlined regions in SEQ ID NO: 4 in Table 1 arebelieved to be especially important for catalysis in thethaxtomin-reducing reactions disclosed herein (see “A” and “B” α-helicesin FIG. 6 , to which these underlined portions correspond, whichcorrespond, respectively, to the amino acid sequences indicated by thedouble-underline and dashed underline in the sequence shown above) andto SEQ ID NO: 5 and SEQ ID NO: 6. As used herein, “putative catalyticdomain A” and “putative catalytic domain B” refer to SEQ ID NO: 5 andSEQ ID NO: 6, respectively.

In some embodiments, putative catalytic domain A has from about 50% toabout 99% sequence identity to the amino acid sequence identified by SEQID NO: 5, or has about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or about 99% sequence identity to SEQ ID NO: 5, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, putative catalytic domain A isidentical to SEQ ID NO: 5.

In some embodiments, putative catalytic domain B has from about 50% toabout 99% sequence identity to the amino acid sequence identified by SEQID NO: 6, or has about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or about 99% sequence identity to SEQ ID NO: 6, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, putative catalytic domain B isidentical to SEQ ID NO: 6.

In any of these aspects, nitroreductases that have amino acid sequenceswith substantial homology or sequence identity with SEQ ID NO: 5 and SEQID NO: 6 within the overall amino acid sequence for the nitroreductasesare especially effective in the methods and processes disclosed herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. A variant of apolynucleotide that is useful as for producing a nitroreductase enzymewill retain the ability to reduce nitro groups in thaxtomins and relatedmolecules and, in some embodiments, thereby control a pathogenicbacterium of interest. As used herein, a “native” polynucleotide orpolypeptide comprises a naturally occurring nucleotide sequence or aminoacid sequence, respectively. For polynucleotides, conservative variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the amino acid sequence of one of the polypeptides employedin the present disclosure. Variant polynucleotides also includesynthetically derived polynucleotide, such as those generated, forexample, by using site-directed mutagenesis, but continue to retain thedesired activity. Generally, variants of a particular polynucleotide ofthe present disclosure (i.e., a nitroreductase gene) will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Any region of the polynucleotide of the present disclosure (e.g., thenitroreductase gene) can be used for comparing or determining sequenceidentity for other polynucleotide sequences useful herein. In oneaspect, sequence identity can be determined based on the regions of thepolynucleotide sequences corresponding to particular domains of theprotein encoded by the polynucleotide, or any combination thereof. Inone aspect, sequence identity can be to about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 consecutive nucleotidesfrom nucleotides 1-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200,175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400,375-425, 400-450, 425-475, 450-500, 475-525, 500-550, 525-575, 550-600,575-625, 600-650, and/or 625-663 of the polynucleotide sequencedisclosed herein.

Variants of a particular polynucleotide of the present disclosure (i.e.,the reference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of polynucleotidesemployed in the present disclosure is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.” As used herein, “reference sequence”is a defined sequence used as a basis for sequence comparison. Areference sequence may be a subset or the entirety of a specifiedsequence; for example, as a segment of a full-length cDNA or genesequence, or the complete cDNA or gene sequence. As used herein,“comparison window” makes reference to a contiguous and specifiedsegment of a polynucleotide sequence, wherein the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, California).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Protein Variants

As discussed herein, numerous variants of the nitroreductase protein areknown and herein contemplated. In addition, to the known functionalnitroreductase strain variants there are derivatives of thenitroreductase proteins which also function in the disclosed methods andcompositions. Protein variants and derivatives are well understood tothose of skill in the art and in can involve amino acid sequencemodifications. For example, amino acid sequence modifications typicallyfall into one or more of three classes: substitutional, insertional ordeletional variants. Insertions include amino and/or carboxyl terminalfusions as well as intrasequence insertions of single or multiple aminoacid residues. Insertions ordinarily will be smaller insertions thanthose of amino or carboxyl terminal fusions, for example, on the orderof one to four residues. Immunogenic fusion protein derivatives, such asthose described in the examples, are made by fusing a polypeptidesufficiently large to confer immunogenicity to the target sequence bycross-linking in vitro or by recombinant cell culture transformed withDNA encoding the fusion. Deletions are characterized by the removal ofone or more amino acid residues from the protein sequence. Typically, nomore than about from 2 to 6 residues are deleted at any one site withinthe protein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 2 and 3 and are referred to as conservative substitutions.

TABLE 2 Amino Acid Abbreviations Amino Acid Abbreviations Alanine Ala AAllosoleucine AIle Arginine Arg R Asparagine Asn N Aspartic Acid Asp DCysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KPhenylalanine Phe F Proline Pro P Pyroglutamic Acid pGlu Serine Ser SThreonine Thr T Tyrosine Tyr Y Tryptophan Trp W Valine Val V

TABLE 3 Amino Acid Substitutions Original Exemplary ConservativeSubstitutions Residue (Others Known in the Art) Ala Ser Arg Lys; Gln AsnGln; His Asp Glu Cys Ser Gln Asn; Lys Glu Asp Gly Pro His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Ley; Tyr SerThr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table3, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions that,in general, are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; (d) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having a side chain, e.g., glycine,in this case; or (e) by increasing the number of sites for sulfationand/or glycosylation.

The replacement of one amino acid residue with another that isbiologically and/or chemically similar is known to those skilled in theart as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, areaccomplished for example by deleting one of the basic residues orsubstituting one with glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 1 sets forth a particular sequence of anitroreductase gene and SEQ ID NO: 4 sets forth a particular sequence ofa nitroreductase protein. Specifically disclosed are variants of theseand other proteins herein disclosed which have at least, 70% or 75% or80% or 85% or 90% or 95% homology to the stated sequence. Those of skillin the art readily understand how to determine the homology of twoproteins. For example, the homology can be calculated after aligning thetwo sequences so that the homology is at its highest level.

Active Fragments of Nitroreductase Sequences

Fragments and variants of the nitroreductase polynucleotides andpolypeptides can be employed in the methods and compositions disclosedherein. By “fragment” is intended a portion of the polynucleotide or aportion of the amino acid sequences and, hence, protein encoded thereby.Fragments of a polynucleotide can encode protein fragments that retainnitroreductase activity. Thus, fragments of a nucleotide sequence canrange from at least about 20 nucleotides, about 50 nucleotides, about100 nucleotides, up to the full-length polynucleotide encoding thenitroreductase polypeptides.

A fragment of a nitroreductase polypeptide that encodes a biologicallyactive portion of a nitroreductase polypeptide will encode at least 25,50, 75, 100, 125, 150, 175, 200, or 220 contiguous amino acids, or up tothe total number of amino acids present in a full length nitroreductasepolypeptide as set forth in, for example, SEQ ID NO: 4 or an activevariant or fragment thereof.

In other embodiments, a fragment of a nitroreductase polynucleotide thatencodes a biologically active portion of a nitroreductase polypeptidewill encode a region of the polypeptide that is sufficient to form thenitroreductase residue geometry (i.e., putative catalytic domain Aand/or putative catalytic domain B) as set forth in SEQ ID Nos. 5 and 6.

In some embodiments, biologically active variants of a nitroreductasepolypeptide (and the polynucleotide encoding the same) will have apercent identity across their full length of at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the polynucleotide of SEQ ID NO: 1 or the polypeptide of SEQID NO: 4 as determined by sequence alignment programs and parametersdescribed elsewhere herein.

In other embodiments, biologically active variants of a nitroreductasepolypeptide (and the polynucleotide encoding the same) will have atleast a percent similarity score of at least 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater to either SEQ IDNO: 1 (for polynucleotides) or SEQ ID NO: 4 (for polypeptides).

The nitroreductase polypeptides and the active variants and fragmentsthereof may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions and throughrational design modeling. Methods for such manipulations are generallyknown in the art. For example, amino acid sequence variants andfragments of the nitroreductase polypeptides can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference in their entirety.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be optimal.

Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and optimallywill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444.

In various aspects, the putative catalytic domains (SEQ ID Nos: 5 and 6)can have one or more amino acids changed by site-directed mutagenesis ofthe correspondencing nucleotide sequence in SEQ ID NO: 4. That is, theamino acid sequence of the putative catalytic domains (SEQ ID Nos: 5 and6) can be modified to modulate or attenuate nitroreductase activityand/or specificity. For instance, one or more amino acids in one or bothcatalytic domains can be specifically mutated based on analysis ofhomologous and/or orthologous catalytic domains, and the mutantsscreened for activity and specificity. In a further embodiment, theputative catalytic domains (SEQ ID Nos: 5 and 6) can have a level ofhomology to the unmutated sequence that is about 40%, 75% 50%, 55%, 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acidhomology.

Non-limiting examples of nitroreductases and active fragments andvariants thereof are provided herein and can include nitroreductasescomprising an active site having catalytic residue geometries shaped bythe residues forming putative catalytic domain A and putative catalyticdomain B as defined elsewhere herein, or having substantially similarcatalytic residue geometries, and further comprising amino acidsequences having at least 40%, 75% 50%, 55%, 60%, 65%, 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% percent identity to any one of SEQ IDNOs: 5 and 6, wherein the polypeptide has nitroreductase activity. In analternative aspect, putative catalytic domain A can have 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid matches compared to SEQID NO: 5. In another aspect, putative catalytic domain B can have 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 amino acid matchescompared to SEQ ID NO: 6.

In other embodiments, the nitroreductases and active fragments andvariants thereof are provided herein and can include a nitroreductasethat comprises an active site having catalytic residue geometries shapedby the residues forming putative catalytic domain A and putativecatalytic domain B as defined elsewhere herein, or having substantiallysimilar catalytic residue geometries, and further comprising amino acidsequences having percent similarity scores of at least 40%, 75% 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater to anyone of SEQ ID NOs: 5 or 6, wherein the polypeptide has nitroreductaseactivity.

Plants Resistant to Thaxtomins

Disclosed herein are compositions and methods for protecting plants froma pathogenic microorganism, such as a Streptomyces species or strain, orinducing resistance in a plant to a pathogenic microorganism, such as aStreptomyces species or strain.

As used herein, “Streptomyces plant pest” is used to refer to any memberof the Streptomyces genus. Accordingly, the compositions and methodsdisclosed herein are also useful in protecting plants against anyStreptomyces plant best including, but not limited to, Streptomycesscabies, Streptomyces turgidiscabies, Streptomyces acidiscabies,Streptomyces luridiscabiei, Streptomyces puniciscabiei, Streptomycesnieviscabei, Streptomyces ipomoea, and related organisms.

Those skilled in the art will recognize that not all compositions areequally effective against all pests. Disclosed compositions, includingthe nitroreductase enzymes disclosed herein, display activity againstphytotoxic pathogenic microorganisms, including microorganisms thatsecrete thaxtomins.

In one aspect, disclosed herein are plants resistant to thaxtomins. Inanother aspect, the plants incorporate one or more exogenous genes for anitroreductase enzyme. In another aspect, the exogenous genes have beenintroduced by any common genetic engineering or genome editing techniqueknown in the art including, but not limited to, use of a biolisticdevice, a Ti-plasmid, an engineered nuclease (meganucleases, zinc fingernucleases, transcription activator-like effector-based nucleases, and/orCRISPR/Cas9). In still another aspect, the genes are incorporated intothe nuclear DNA of the plant, exist on a plasmid (e.g., a Ti-plasmid)inside the plant cell, or are incorporated into mitochondrial or plastidDNA. In any of the above aspects, incorporation of the one or morenitroreductase genes results in production of the nitroreductase enzymeby the plant. In a still further aspect, production of thenitroreductase enzyme by the plant provides the plant with resistance tothaxtomins. In one aspect, production of the nitroreductase enzymeprovides the plant with resistance to thaxtomins produced by pests orpathogens. In another aspect, production of the nitroreductase enzymeprovides the plant with resistance to thaxtomins incorporated intoagricultural compositions such as, for example, herbicides. In stillanother aspect, production of the nitroreductase enzyme provides theplant with simultaneous resistance to both thaxtomins produced by pestsor pathogens and thaxtomins included in agricultural compositions.

In one aspect, crop plants and/or desirable plants expressingnitroreductase enzymes can be planted in a field that has been treatedprior to planting with thaxtomins to control weeds or other unwantedplants. In another aspect, a field wherein plants expressingnitroreductase enzymes are growing can be treated with thaxtomins. Inone aspect, treating the field with thaxtomins controls and/or killsweeds or other unwanted plants through interference with cellulosesynthesis while not damaging the plants expressing nitroreductase, sincethe nitroreductase expressed by the crop plants or desirable plantsinactivates the thaxtomins.

In one aspect, disclosed herein is a plant cell with stably integrated,recombinant DNA including a nucleotide sequence that encodes anitroreductase protein. In a further aspect, the plant cell alsoincludes a heterologous promoter that is functional in plant cells andthat is operably linked to the nucleotide sequence that encodes thenitroreductase protein. In still another aspect, the nucleotide sequencethat encodes the nitroreductase protein is isolated from Haemophilusinfluenzae, Actinobacillus indolicus, Avibacterium paragallinarum,Mannheimia succiniproducens, Staphylococcus arlettae, Actinobacillussuccinogenes, or Arcobacter molloscorum. In another aspect, thenucleotide sequence that encodes the nitroreductase protein is known asNfsB and/or has at least 90% sequence identity with SEQ ID NO: 1, or atleast 95% sequence identity with SEQ ID NO: 1, or at least 97% sequenceidentity with SEQ ID NO: 1. In any of these aspects, the gene can be acDNA copy of the natural gene (i.e., consisting only of exons and/orcoding sequences and with introns, if any, and 5′ and/or 3′ untranslatedregions having been removed).

In another aspect, disclosed herein are transgenic plants, seeds, and/orcalluses having a plurality of the plant cells described above as wellas their progeny, wherein their progeny plants also include thenucleotide sequence encoding a nitroreductase protein. In one aspect,the transgenic plants and progeny plants are potato plants, beet plants,carrot plants, parsnip plants, radish plants, rutabaga plants, turnipplants, or sweet potato plants. In still another aspect, disclosedherein are vegetables harvested from the transgenic plants.

Exemplary methods for making plants resistant to thaxtomins are providedbelow.

In another aspect, disclosed herein is a method for reducing a plantdamage due to a plant pathogenic organism including providing to a plantor soil before or after introduction of a seed, bulb, tuber, bud, stem,corm, plant part, or a plant, a composition including the DNA constructor expression cassette disclosed herein, wherein the DNA construct orexpression cassette includes a nucleotide sequence encoding anitroreductase protein. In another aspect, disclosed herein is a methodfor reducing the damage caused by common scab of potato to the roots ofa plant including providing to a plant or soil before or afterintroduction of a seed, bulb, tuber, bud, stem, corm, plant part, or aplant, a composition including the DNA construct, expression cassette,or bacterial or plant host cell disclosed herein, wherein the DNAconstruct, expression cassette, or host cell includes a nucleotidesequence encoding a nitroreductase protein. In another aspect, disclosedherein is a method for reducing the damage caused by one or morethaxtomins to the roots of a plant including providing to a plant orsoil before or after introduction of a seed, bulb, tuber, bud, stem,corm, plant part, or a plant, a composition including the DNA construct,expression cassette, or bacterial or plant host cell disclosed herein,wherein the DNA construct, expression cassette, or host cell includes anucleotide sequence encoding a nitroreductase protein.

Method of Making a Plant Resistant to Thaxtomin A DNA Constructs

The use of the term “polynucleotide” is not intended to limit thedisclosed polynucleotides to polynucleotides comprising DNA. Those ofordinary skill in the art will recognize that polynucleotides cancomprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the present disclosure also encompass all forms ofsequences including, but not limited to, single-stranded forms,double-stranded forms, hairpins, stem-and-loop structures, and the like.

The polynucleotide encoding the nitroreductase enzyme or in specificembodiments employed in the methods and compositions of the presentdisclosure can be provided in expression cassettes for expression in aplant or organism of interest. It is recognized that genes for multiplenitroreductase enzymes including multiple identical nitroreductaseenzyme genes or multiple nitroreductase enzyme genes targeting differentthaxtomins. In this embodiment, it is recognized that eachnitroreductase enzyme gene can be contained in a single or separatecassette, DNA construct, or vector. As discussed, any means of providingthe polynucleotide encoding the nitroreductase enzyme is contemplated. Aplant or plant cell can be transformed with a single cassette comprisingDNA encoding one or more nitroreductase genes or separate cassettescomprising each nitroreductase genes can be used to transform a plant orplant cell or host cell. Likewise, a plant transformed with onecomponent can be subsequently transformed with the second component. Oneor more nitroreductase genes can also be brought together by sexualcrossing. That is, a first plant comprising one component is crossedwith a second plant comprising the second component. Progeny plants fromthe cross will comprise both components.

The expression cassette can include 5′ and 3′ regulatory sequencesoperably linked to the polynucleotide of the present disclosure.“Operably linked” is intended to mean a functional linkage between twoor more elements. For example, an operable linkage between apolynucleotide of the present disclosure and a regulatory sequence(i.e., a promoter) is a functional link that allows for expression ofthe polynucleotide of the present disclosure. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, by operably linked is intended that thecoding regions are in the same reading frame. The cassette mayadditionally contain at least one additional polynucleotide to becotransformed into the organism. Alternatively, the additionalpolypeptide(s) can be provided on multiple expression cassettes.Expression cassettes can be provided with a plurality of restrictionsites and/or recombination sites for insertion of the polynucleotide tobe under the transcriptional regulation of the regulatory regions. Theexpression cassette may additionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a polynucleotide comprising the nitroreductase geneemployed in the methods and compositions of the present disclosure, anda transcriptional and translational termination region (i.e.,termination region) functional in plants. “Divergent promoters” refersto promoters that are oriented in opposite directions of each other,driving transcription of the one or more nitroreductase genes inopposite directions. In another embodiment, one cassette comprising twoor more nitroreductase genes under the control of two separate promotersin the same orientation is present in a construct. In anotherembodiment, two or more individual cassettes, each comprising at leastone nitroreductase genes under the control of a promoter, are present ina construct in the same orientation.

The regulatory regions (i.e., promoters, transcriptional regulatoryregions, and translational termination regions) and/or thepolynucleotides employed in the present disclosure may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the polynucleotide employed in the presentdisclosure may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide. Asused herein, a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked polynucleotide encodingthe nitroreductase gene, may be native with the plant host, or may bederived from another source (i.e., foreign or heterologous) to thepromoter, the polynucleotide comprising nitroreductase enzyme, the planthost, or any combination thereof. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acids Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the presentdisclosure. The polynucleotide encoding the nitroreductase gene can becombined with constitutive, tissue-preferred, or other promoters forexpression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include thosetaught in, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

An inducible promoter, for instance, a pathogen-inducible promoter couldalso be employed. Such promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, β-1,3-glucanase, chitinase,etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol.89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon(1985) Plant Mol. Viral. 4:111-116. See also WO 99/43819, hereinincorporated by reference.

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the present disclosure. Such wound-inducible promotersinclude potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-la promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters includeYamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teen et al. (1989) usedgene fusion to lacZ to show that the Agrobacterium T-DNA gene encodingoctopine synthase is especially active in the epidermis of the root tipand that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptll (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

In an embodiment, the plant-expressed promoter is a vascular-specificpromoter such as a phloem-specific promoter. A “vascular-specific”promoter, as used herein, is a promoter which is at least expressed invascular cells, or a promoter which is preferentially expressed invascular cells. Expression of a vascular-specific promoter need not beexclusively in vascular cells, expression in other cell types or tissuesis possible. A “phloem-specific promoter” as used herein, is aplant-expressible promoter which is at least expressed in phloem cells,or a promoter which is preferentially expressed in phloem cells.

Expression of a phloem-specific promoter need not be exclusively inphloem cells, expression in other cell types or tissues, e.g., xylemtissue, is possible. In one embodiment of this disclosure, aphloem-specific promoter is a plant-expressible promoter at leastexpressed in phloem cells, wherein the expression in non-phloem cells ismore limited (or absent) compared to the expression in phloem cells.Examples of suitable vascular-specific or phloem-specific promoters inaccordance with this disclosure include but are not limited to thepromoters selected from the group consisting of: the SCSV3, SCSV4,SCSV5, and SCSV7 promoters (Schunmann et al. (2003) Plant FunctionalBiology 30:453-60; the roIC gene promoter of Agrobacteriumrhizogenes(Kiyokawa et al. (1994) Plant Physiology 104:801-02;Pandolfini et al. (2003) BioMedCentral (BMC) Biotechnology 3:7,(www.biomedcentral.com/1472-6750/317); Graham et al. (1997) Plant Mol.Biol. 33:729-35; Guivarc'h et al. (1996); Almon et al. (1997) PlantPhysiol. 115:1599-607; the rolA gene promoter of Agrobacteriumrhizogenes (Dehio et al. (1993) Plant Mol. Biol. 23:1199-210); thepromoter of the Agrobacterium tumefaciens T-DNA gene 5 (Korber et al.(1991) EMBO J. 10:3983-91); the rice sucrose synthase RSs1 gene promoter(Shi et al. (1994) J. Exp. Bot. 45:623-31); the CoYMV or Commelinayellow mottle badnavirus promoter (Medberry et al. (1992) Plant Cell4:185-92; Zhou et al. (1998) Chin. J. Biotechnol. 14:9-16); the CFDV orcoconut foliar decay virus promoter (Rohde et al. (1994) Plant Mol.Biol. 27:623-28; Hehn and Rhode (1998) J. Gen. Viral. 79:1495-99); theRTBV or rice tungro bacilliform virus promoter (Yin and Beachy (1995)Plant J. 7:969-80; Yin et al. (1997) Plant J. 12:1179-80); the peaglutamin synthase GS3A gene (Edwards et al. (1990) Proc. Natl. Acad.Sci. USA 87:3459-63; Brears et al. (1991) Plant J. 1:235-44); the invCD111 and inv CD141 promoters of the potato invertase genes (Hedley etal. (2000) J. Exp. Botany 51:817-21); the promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al. (1991) Proc. Natl. Acad. Sci. USA 88:5212-16); theVAHOXI promoter region (Tornero et al. (1996) Plant J. 9:639-48); thepea cell wall invertase gene promoter (Zhang et al. (1996) PlantPhysiol. 112:1111-17); the promoter of the endogenous cotton proteinrelated to chitinase of US published patent application 20030106097, anacid invertase gene promoter from carrot (Ramloch-Lorenz et al. (1993)The Plant J. 4:545-54); the promoter of the sulfate transportergeneSultr1; 3 (Yoshimoto et al. (2003) Plant Physiol. 131:1511-17); apromoter of a sucrose synthase gene (Nolte and Koch (1993) PlantPhysiol. 101:899-905); and the promoter of a tobacco sucrose transportergene (Kuhn et al. (1997) Science 275-1298-1300).

Possible promoters also include the Black Cherry promoter for PrunasinHydrolase (PH DL1.4 PRO) (U.S. Pat. No. 6,797,859), Thioredoxin Hpromoter from cucumber and rice (Fukuda A et al. (2005). Plant CellPhysiol. 46(11):1779-86), Rice (RSs1) (Shi, T. Wang et al. (1994). J.Exp. Bot. 45(274): 623-631) and maize sucrose synthase-1 promoters(Yang., N-S. et al. (1990) PNAS 87:4144-4148), PP2 promoter from pumpkinGuo, H. et al. (2004) Transgenic Research 13:559-566), At SUC2 promoter(Truemit, E. et al. (1995) Planta 196(3):564-70., At SAM-1(S-adenosylmethionine synthetase) (Mijnsbrugge KV. et al. (1996) PlantCell. Physiol. 37(8): 1108-1115), and the Rice tungro bacilliform virus(RTBV) promoter (Bhattacharyya-Pakrasi et al. (1993) Plant J.4(1):71-79).

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP from Evrogen, see, Bolte et al. (2004)J. Cell Science 117:943-54). For additional selectable markers, seegenerally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Nall.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used with the disclosed polynucleotides,constructs, vectors, methods, and compositions.

In one aspect, disclosed herein is a DNA construct having a nucleotidesequence encoding a nitroreductase protein. In a further aspect, the DNAconstruct further includes a heterologous promoter that is functional inplant cells and that is operably linked to the nucleotide sequence thatencodes the nitroreductase protein. In still another aspect, thenucleotide sequence that encodes the nitroreductase protein is isolatedfrom Haemophilus influenzae, Actinobacillus indolicus, Avibacteriumparagallinarum, Mannheimia succiniproducens, Staphylococcus arlettae,Actinobacillus succinogenes, Arcobacter molloscorum, or a relatedorganism. In yet another aspect, the nitroreductase protein can be NfsBand/or the sequence that encodes it can have at least 90% sequenceidentity with SEQ ID NO: 1, at least 95% sequence identity with SEQ IDNO: 1, or at least 97% sequence identity with SEQ ID NO: 1. In someaspects, the nucleotide sequence encoding a nitroreductase protein is acDNA copy of a natural gene with introns removed.

In another aspect, disclosed herein is an expression cassette containingthe DNA construct having a nucleotide sequence encoding a nitroreductaseprotein. In a further aspect, the nucleotide sequence in the DNAconstruct is operably linked to a heterologous promoter.

In one aspect, disclosed herein is a plant chromosomal DNA segment thatincludes a recombinant polynucleotide flanked by native plant DNA,wherein the polynucleotide provides for expression of at least anitroreductase protein. In another aspect, the plant chromosomal DNAsegment includes a recombinant DNA construct for expressing anitroreductase protein wherein the protein includes contiguous aminoacids having at least 90% sequence identity to SEQ ID NO: 4, at least95% sequence identity to SEQ ID NO: 4, or at least 97% sequence identityto SEQ ID NO: 4. Also disclosed herein are transgenic plant cellsincluding the disclosed plant chromosomal DNA segment. In some aspects,the nitroreductase protein includes portions having SEQ ID NO: 5 and SEQID NO: 6, herein referred to as putative catalytic domain A and putativecatalytic domain B, respectively, or at least 70% sequence identitythereto, at least 75% sequence identity thereto, at least 80% sequenceidentity thereto, or at least 85% sequence identity thereto.

In some embodiments, putative catalytic domain A has from about 50% toabout 99% sequence identity to the amino acid sequence identified by SEQID NO: 5, or has about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or about 99% sequence identity to SEQ ID NO: 5, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, putative catalytic domain A isidentical to SEQ ID NO: 5.

In some embodiments, putative catalytic domain B has from about 50% toabout 99% sequence identity to the amino acid sequence identified by SEQID NO: 6, or has about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or about 99% sequence identity to SEQ ID NO: 6, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, putative catalytic domain B isidentical to SEQ ID NO: 6.

In one aspect, disclosed herein is a method for improving resistance toat least one thaxtomin in a crop plant line, the method includingproviding in the genome of the crop plant the disclosed chromosomal DNAsegment. In a further aspect, the thaxtomin is secreted by a pathogenicmicroorganism such as, for example, Streptomyces scabies, Streptomycesturgidiscabies, Streptomyces acidiscabies, Streptomyces luridiscabiei,Streptomyces puniciscabiei, Streptomyces nieviscabei, Streptomycesipomoea, or a combination thereof.

In an alternative aspect, the thaxtomin can be exogenously applied. Instill another aspect, the method improves resistance to bothmicrobially-secreted and exogenously applied thaxtomins simultaneously.In another aspect, the thaxtomin can be thaxtomin A, thaxtomin B,thaxtomin C, and/or thaxtomin D.

Plants, Plant Parts, and Methods of Introducing Sequences into Plants

In one embodiment, the methods of the present disclosure involveintroducing a polynucleotide into a plant. “Introducing” is intended tomean presenting to the plant the polynucleotide in such a manner thatthe sequence gains access to the interior of a cell of the plant. Themethods of the present disclosure do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotides into plants are known inthe art including, but not limited to, stable transformation methods,transient transformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320 334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602 5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717 2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923926); and Lec1 transformation (WO 00/28058). Also see Weissinger et al.(1988) Ann. Rev. Genet. 22:421 477; Sanford et al. (1987) ParticulateScience and Technology 5:27 37 (onion); Christou et al. (1988) PlantPhysiol. 87:671 674 (soybean); McCabe et al. (1988) Bio/Technology 6:923926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736 740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305 4309(maize); Klein et al. (1988) Biotechnology 6:559 563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440 444 (maize); Fromm et al. (1990) Biotechnology 8:833 839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the nitroreductase-encoding polynucleotidesequences of the present disclosure can be provided to a plant using avariety of transient transformation methods. Such transienttransformation methods include, but are not limited to, the introductionof the protein or variants and fragments thereof directly into the plantor the introduction of the transcript into the plant. Such methodsinclude, for example, microinjection or particle bombardment. See, forexample, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura etal. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad.Sci. 91: 2176-2180 and Hush et al. (1994) J. Cell Sci. 107:775-784, allof which are herein incorporated by reference. Alternatively,polynucleotides can be transiently transformed into the plant usingtechniques known in the art. Such techniques include viral vectorsystems and the precipitation of the polynucleotide in a manner thatprecludes subsequent release of the DNA. Thus, the transcription fromthe particle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

In other embodiments, the polynucleotide of the present disclosure maybe introduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the present disclosure within a viral DNA or RNAmolecule. Further, it is recognized that promoters of the presentdisclosure also encompass promoters utilized for transcription by viralRNA polymerases. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Mol. Biotechnol. 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the present disclosure can be contained intransfer cassette flanked by two non-recombinogenic recombination sites.The transfer cassette is introduced into a plant having stablyincorporated into its genome a target site which is flanked by twonon-recombinogenic recombination sites that correspond to the sites ofthe transfer cassette. An appropriate recombinase is provided and thetransfer cassette is integrated at the target site. The polynucleotideof interest is thereby integrated at a specific chromosomal position inthe plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, provided herein are transformed seed (also referred toas “transgenic seed”) having a polynucleotide of the present disclosure,for example, an expression cassette of the present disclosure, stablyincorporated into their genome.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the presentdisclosure, provided that these parts comprise the introducedpolynucleotides.

The compositions, methods, constructs, and polynucleotides may be usedfor transformation of any plant species including, but not limited to,potato (Solanum tuberosum), beet (Beta vulgaris), carrot (Daucuscarota), parsnip (Pastinaca sativa), radish (Raphanus raphanistrum),rutabaga (Brassica napobrassica), turnip (Brassica rapa subsp. Rapa),and/or sweet potato (Ipomoea batatas).

In one aspect, disclosed herein is a host cell containing the DNAconstruct and/or expression cassette disclosed herein. In some aspects,the host cell can be a bacterial cell. In another aspect, disclosedherein is a plant cell having stably incorporated into its genome aheterologous polynucleotide comprising a nucleotide sequence encoding anitroreductase protein, wherein the nitroreductase protein istranscribed and translated from a nucleotide sequence having at least90% sequence identity with SEQ ID NO: 1, at least 95% sequence identitywith SEQ ID NO: 1, or at least 97% sequence identity with SEQ ID NO: 1or a variant or fragment thereof, and wherein the nucleotide sequenceencoding the nitroreductase protein, when transcribed and translated,produces a protein capable of reducing a nitro group on a thaxtomin.

Agricultural Products from Transgenic Plants

In one aspect, disclosed herein are agricultural products harvested fromtransgenic plants produced as described herein. In one aspect, theagricultural products are resistant to diseases such as common scab ofpotato and related conditions, both before and after harvest. In anotheraspect, the agricultural products are resistant to cellular damagecaused by thaxtomins, both before and after harvest. In still anotheraspect, the agricultural products are resistant to exogenously-appliedagricultural products containing thaxtomins (i.e., herbicides), or areresistant to thaxtomins secreted by pests and/or pathogens, or both. Ina further aspect, the agricultural products can be potatoes, sweetpotatoes, beets, carrots, parsnips, radishes, rutabagas, turnips, andother common starchy root vegetables susceptible to degradation bythaxtomins.

In any of the above aspects, the agricultural products are safe forhuman and animal consumption and can be used for any purpose commonlyknown in the art including food, animal feed, production of natural dyes(e.g., from beets or carrots), production of sugars (e.g., from beets),production of calluses for tissue culture, extraction of starches (e.g.,from potato), or the like.

In one aspect, the agricultural products include or are composed oftransgenic plant cells capable of expressing one or more nitroreductaseenzymes, wherein the nitroreductase enzyme converts one or morethaxtomin nitro groups to an amino group.

Method for Scaling Production of 4-Amino Thaxtomins

In one aspect, disclosed herein is a method for scaling production of4-amino thaxtomins for use in research and other applications. In oneaspect, the method includes the following steps:

-   -   (a) culturing in a culture medium an organism known to produce        one or more thaxtomins;    -   (b) using the culture medium as a crude extract containing one        or more thaxtomins;    -   (c) culturing recombinant cells incorporating one or more        nitroreductase genes including inducing enzyme production;    -   (d) lysing the recombinant cells to release the nitroreductase        proteins and form a cell lysate incorporating the same;    -   (e) contacting the crude extract containing one or more        thaxtomins with a composition containing the lysed recombinant        cells to create a mixture;    -   (f) incubating the mixture such that the nitroreductase proteins        reduce the nitro groups on the thaxtomins to form 4-amino        thaxtomins; and    -   (g) purifying the 4-amino thaxtomins.

In a further aspect, the reaction can be performed with from about 50 toabout 250 mL of reaction mixture, or with about 50, 75, 100, 125, 150,175, 200, 225, or about 250 mL of reaction mixture, or a combination ofany of the foregoing values, or a range encompassing any of theforegoing values. In one aspect, the reaction is performed with about100 mL of reaction mixture. In another aspect, the reaction mixtureincludes at least the following components. In one aspect, the reactionmixture includes from about 5 to about 10 mL of cell lysateincorporating nitroreductase proteins, or about 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, or about 10 mL of cell lysate, or a combination of anyof the foregoing values, or a range encompassing any of the foregoingvalues. In one aspect, 7.5 mL of cell lysate incorporatingnitroreductase proteins is used. In another aspect, from about 1 toabout 10 mL of crude extract containing thaxtomins is included in thereaction mixture, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 mL ofcrude extract, or a combination of any of the foregoing values, or arange encompassing any of the foregoing values. In one aspect, 2 mL ofcrude extract containing thaxtomins is used. In a further aspect, thethaxtomins can be suspended in or extracted into DMSO at a concentrationof from 15 to 30 mg/mL, or at about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or about 30 mg/mL, or a combination of any ofthe foregoing values, or a range encompassing any of the foregoingvalues. In another aspect, the crude extract contains about 21.9 mg/mLof thaxtomins in DMSO. In yet another aspect, the crude extract containsfrom about 60 to about 80% thaxtomin A, or about 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80%thaxtomin A, or a combination of any of the foregoing values, or a rangeencompassing any of the foregoing values. In one aspect, the crudeextract contains about 69% thaxtomin A, with the remainder includingother thaxtomins. In still another aspect, the crude extract includes abuffer such as, for example, 50 mM Tris-HCl buffer in an amountsufficient to maintain the reaction mixture at a pH of 8.0. In anotheraspect, the reaction mixture includes a flavin mononucleotide (FMN)solution. In one aspect, about 0.5 mL of a 50 mM FMN solution is addedto the reaction mixture. In another aspect, the reaction mixtureincludes an NADP solution. In a further aspect, about 2 mL of a 50 mMNADP solution is added to the reaction mixture. In still another aspect,the reaction mixture can include other components such as, for example,glutamate dehydrogenase (GDH, 1 mL of an 0.75 mM solution), glucose (1.5mL of a 40% solution), or other components as described herein or knownin the art.

In one aspect, the reaction mixture once formed is incubated at atemperature of from about 30 to about 50° C., or at about 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, orabout 50° C., or a combination of any of the foregoing values, or arange encompassing any of the foregoing values. In one aspect, thereaction mixture is incubated at about 37° C. In another aspect, thereaction mixture is incubated for a period of from about 2 to about 10hours, or for about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 hours, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, the reaction mixture isincubated for about 6 hours.

In any of the above aspects, the reaction can be terminated by adding asolvent such as, for example, ethyl acetate. In one aspect, the volumeof ethyl acetate added to the reaction mixture is equal to the totalvolume of the reaction mixture. In a further aspect, the mixture can beextracted one, two, three, or four times with ethyl acetate. In any ofthese aspects, the organic layer can from each extraction can becombined, washed with water, dried, and evaporated. In still anotheraspect, the residue left after evaporation can be purified by a methodknown in the art such as, for example, high pressure liquidchromatography (HPLC). In still another aspect, HPLC fractionscontaining the product of interest can be combined and lyophilized toyield solid 4-amino thaxtomins.

Agricultural Compositions Containing Thaxtomins Formulations

The present disclosure also concerns agricultural compositionscomprising or consisting essentially of an active compound such as, forexample, a thaxtomin, as described herein in combination with a suitablecarrier (e.g., an agricultural carrier) or adjuvant (e.g., andagricultural adjuvant). In some aspects, the disclosed agriculturalcompositions are useful as herbicidal compositions for use withcommercially useful plants and crops, including crops of useful plants.

The agricultural composition of the present disclosure, e.g., adisclosed herbicidal composition, can contain from 0.1 to 99% by weight,e.g., from 0.1 to 95% by weight, of a disclosed compound, 99.9 to 1% byweight, e.g., 99.8 to 5% by weight, of a solid or liquid adjuvant, andoptionally from 0 to 25% by weight, e.g., from 0.1 to 25% by weight, ofa surfactant.

The agricultural composition of the present disclosure, e.g., adisclosed herbicidal composition, can contain from 0.1 to 99% by weight,e.g., from 0.1 to 95% by weight, of a disclosed compound, 99.9 to 1% byweight, e.g., 99.8 to 5% by weight, of a solid or liquid carrier, andoptionally from 0 to 25% by weight, e.g., from 0.1 to 25% by weight, ofa surfactant.

Suitably, an agricultural composition of the present disclosure, e.g., adisclosed herbicidal composition, can be applied at any suitabledevelopmental stage of the crop or plant. Rates and frequency of use ofthe formulations are those conventionally used in the art and factorssuch as the developmental stage of the plant and on the location, timingand application method, and density and development stage of theundesirable plant, e.g., a weed. Advantageous rates of application canrange from 5 g to 2 kg of active ingredient (a.i.) per hectare (ha),preferably from 10 g to 1 kg a.i./ha, most preferably from 20 g to 600 ga.i./ha. When used as seed drenching agent, convenient rates ofapplication are from 10 mg to 1 g of active substance per kg of seeds.

In practice, as indicated above, an agricultural composition of thepresent disclosure, e.g., a disclosed herbicidal composition, can beapplied as a formulation containing the various adjuvants and carriersknown to or used in the industry. They may thus be formulated asgranules, as wettable or soluble powders, as emulsifiable concentrates,as coatable pastes, as dusts, as flowables, as solutions, as suspensionsor emulsions, or as controlled release forms such as microcapsules.These formulations are described in more detail below and may contain aslittle as about 0.5% to as much as about 95% or more by weight of theactive ingredient. The optimum amount will depend on formulation,application equipment and nature of the plant to be treated. Inaddition, the disclosed agricultural compositions, e.g., an herbicidalcomposition, can optionally further comprise conventional additives suchas surfactants, drift reduction agents, safeners, solubility enhancingagents, thickening agents, flow enhancers, foam-moderating agents,freeze protectants, UV protectants, preservatives, antimicrobials,and/or other additives that are necessary or desirable to improve theperformance, crop safety, or handling of the composition.

Suitable agricultural adjuvants and carriers that are useful informulating the compositions of the present disclosure in theformulation types described above are well known to those skilled in theart. Other adjuvants commonly utilized in agricultural compositionsinclude crystallization inhibitors, viscosity modifiers, suspendingagents, spray droplet modifiers, pigments, antioxidants, foaming agents,anti-foaming agents, light-blocking agents, compatibilizing agents,antifoam agents, sequestering agents, neutralizing agents and buffers,corrosion inhibitors, dyes, odorants, spreading agents, penetrationaids, micronutrients, emollients, lubricants, sticking agents, and thelike. Suitable examples of the different classes are found in thenon-limiting list below.

Exemplary agriculturally acceptable adjuvants include, but are notlimited to, antifreeze agents, antifoam agents, compatibilizing agents,sequestering agents, neutralizing agents and buffers, corrosioninhibitors, colorants, odorants, penetration aids, wetting agents,spreading agents, dispersing agents, thickening agents, freeze pointdepressants, antimicrobial agents, crop oil, safeners, adhesives (forinstance, for use in seed formulations), surfactants, protectivecolloids, emulsifiers, tackifiers, and mixtures thereof. Exemplaryagriculturally acceptable adjuvants include, but are not limited to,crop oil concentrate (mineral oil (85%)+emulsifiers (15%)) or less,nonylphenol ethoxylate or less, benzylcocoalkyldimethyl quaternaryammonium salt or less, blend of petroleum hydrocarbon, alkyl esters,organic acid, and anionic surfactant or less, C9-C11 alkylpolyglycosideor less, phosphate alcohol ethoxylate or less, natural primary alcohol(C12-C16) ethoxylate or less, di-sec-butylphenol EO-PO block copolymeror less, polysiloxane-methyl cap or less, nonylphenol ethoxylate+ureaammonium nitrate or less, emulsified methylated seed oil or less,tridecyl alcohol (synthetic) ethoxylate (8 EO) or less, tallow amineethoxylate (15 EO) or less, and PEG(400) dioleate-99.

In some aspects, the additive is a safener that is an organic compoundleading to better crop plant compatibility when applied with anherbicide. In some aspects, the safener itself is herbicidally active.In some, the safener acts as an antidote or antagonist in the cropplants and can reduce or prevent damage to the crop plants. Exemplarysafeners include, but are not limited to, AD-67 (MON 4660), benoxacor,benthiocarb, brassinolide, cloquintocet (mexyl), cyometrinil,cyprosulfamide, daimuron, dichlormid, dicyclonon, dietholate,dimepiperate, disulfoton, fenchlorazole, fenchlorazole-ethyl, fenclorim,flurazole, fluxofenim, furilazole, harpin proteins, isoxadifen-ethyl,jiecaowan, jiecaoxi, mefenpyr, mefenpyr-diethyl, mephenate, naphthalicanhydride, 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine,4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane, oxabetrinil, R29148, andN-phenyl-sulfonylbenzoic acid amides, as well as agriculturallyacceptable salts and, provided they have a carboxyl group, theiragriculturally acceptable derivatives thereof. In some aspects, thesafener can be cloquintocet or an ester or salt thereof, such ascloquintocet (mexyl). For example, cloquintocet can be used toantagonize harmful effects of the compositions on rice and cereals.

Liquid carriers that can be employed include water, toluene, xylene,petroleum naphtha, crop oil, acetone, methyl ethyl ketone,cyclohexanone, acetic anhydride, acetonitrile, acetophenone, amylacetate, 2-butanone, chlorobenzene, cyclohexane, cyclohexanol, alkylacetates, diacetonalcohol, 1,2-dichloropropane, diethanolamine,p-diethylbenzene, diethylene glycol, diethylene glycol abietate,diethylene glycol butyl ether, diethylene glycol ethyl ether, diethyleneglycol methyl ether, N,N-dimethyl formamide, dimethyl sulfoxide,1,4-dioxane, dipropylene glycol, dipropylene glycol methyl ether,dipropylene glycol dibenzoate, diproxitol, alkyl pyrrolidinone, ethylacetate, 2-ethyl hexanol, ethylene carbonate, 1,1,1-trichloroethane,2-heptanone, alpha pinene, d-limonene, ethylene glycol, ethylene glycolbutyl ether, ethylene glycol methyl ether, gamma-butyrolactone,glycerol, glycerol diacetate, glycerol monoacetate, glycerol triacetate,hexadecane, hexylene glycol, isoamyl acetate, isobornyl acetate,isooctane, isophorone, isopropyl benzene, isopropyl myristate, lacticacid, laurylamine, mesityl oxide, methoxy-propanol, methyl isoamylketone, methyl isobutyl ketone, methyl laurate, methyl octanoate, methyloleate, methylene chloride, m-xylene, n-hexane, n-octylamine,octadecanoic acid, octyl amine acetate, oleic acid, oleylamine,o-xylene, phenol, polyethylene glycol (PEG400), propionic acid,propylene glycol, propylene glycol monomethyl ether, p-xylene, toluene,triethyl phosphate, triethylene glycol, xylene sulfonic acid, paraffin,mineral oil, trichloroethylene, perchloroethylene, ethyl acetate, amylacetate, butyl acetate, methanol, ethanol, isopropanol, and highermolecular weight alcohols such as amyl alcohol, tetrahydrofurfurylalcohol, hexanol, octanol, etc. ethylene glycol, propylene glycol,glycerine, N-methyl-2-pyrrolidinone, and the like. Water is generallythe carrier of choice for the dilution of concentrates.

Suitable solid carriers include talc, titanium dioxide, pyrophylliteclay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth,lime, calcium carbonate, bentonite clay, fuller's earth, cotton seedhulls, wheat flour, soybean flour, pumice, wood flour, walnut shellflour, lignin and the like.

A broad range of surface-active agents are advantageously employed inboth said liquid and solid compositions, especially those designed to bediluted with carrier before application, including surfactants. Theseagents, when used, normally comprise from 0.1% to 15% by weight of theformulation. They can be anionic, cationic, non-ionic or polymeric incharacter and can be employed as emulsifying agents, wetting agents,suspending agents or for other purposes. Typical surface active agentsinclude salts of alkyl sulfates, such as diethanolammonium laurylsulphate; alkylarylsulfonate salts, such as calciumdodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products,such as nonylphenol-C 18 ethoxylate; alcohol-alkylene oxide additionproducts, such as tridecyl alcohol-C 16 ethoxylate; soaps, such assodium stearate; alkylnaphthalenesulfonate salts, such as sodiumdibutylnaphthalenesulfonate; dialkyl esters of sulfosuccinate salts,such as sodium di(2-ethylhexyl) sulfosuccinate; sorbitol esters, such assorbitol oleate; quaternary amines, such as lauryl trimethylammoniumchloride; polyethylene glycol esters of fatty acids, such aspolyethylene glycol stearate; block copolymers of ethylene oxide andpropylene oxide; and salts of mono and dialkyl phosphate esters.

Exemplary surfactants (e.g., wetting agents, tackifiers, dispersants,emulsifiers) include, but are not limited to, the alkali metal salts,alkaline earth metal salts and ammonium salts of aromatic sulfonicacids, for example lignosulfonic acids, phenolsulfonic acids,naphthalenesulfonic acids, and dibutylnaphthalenesulfonic acid, and offatty acids, alkyl- and alkylarylsulfonates, alkyl sulfates, laurylether sulfates and fatty alcohol sulfates, and salts of sulfated hexa-,hepta- and octadecanols, and also of fatty alcohol glycol ethers,condensates of sulfonated naphthalene and its derivatives withformaldehyde, condensates of naphthalene or of the naphthalene sulfonicacids with phenol and formaldehyde, polyoxyethylene octylphenol ether,ethoxylated isooctyl-, octyl- or nonylphenol, alkylphenyl ortributylphenyl polyglycol ether, alkyl aryl polyether alcohols,isotridecyl alcohol, fatty alcohol/ethylene oxide condensates,ethoxylated castor oil, polyoxyethylene alkyl ethers or polyoxypropylenealkyl ethers, lauryl alcohol polyglycol ether acetate, sorbitol esters,lignosulfite waste liquors and proteins, denatured proteins,polysaccharides (e.g., methylcellulose), hydrophobically modifiedstarches, polyvinyl alcohol, polycarboxylates, polyalkoxylates,polyvinyl amine, polyethyleneimine, polyvinylpyrrolidone and copolymersthereof.

Suspension concentrates are aqueous formulations in which finely dividedsolid particles of the active compound are suspended. Such formulationsinclude anti-settling agents and dispersing agents and may furtherinclude a wetting agent to enhance activity as well an anti-foam and acrystal growth inhibitor. In use, these concentrates are diluted inwater and normally applied as a spray to the area to be treated. Theamount of active ingredient, i.e., one or more disclosed compound, mayrange from about 0.5% to about 95% of the concentrate.

Wettable powders are in the form of finely divided particles whichdisperse readily in water or other liquid carriers. The particlescontain the active ingredient retained in a solid matrix. Typical solidmatrices include fuller's earth, kaolin clays, silicas and other readilywet organic or inorganic solids. Wettable powders normally contain about5% to about 95% of the active ingredient plus a small amount of wetting,dispersing or emulsifying agent. Emulsifiable concentrates arehomogeneous liquid compositions dispersible in water or other liquid andmay consist entirely of the active compound with a liquid or solidemulsifying agent, or may also contain a liquid carrier, such as xylene,heavy aromatic naphthas, isophorone and other non-volatile organicsolvents. In use, these concentrates are dispersed in water or otherliquid and normally applied as a spray to the area to be treated. Theamount of active ingredient may range from about 0.5% to about 95% ofthe concentrate.

Granular formulations include both extrudates and relatively coarseparticles and are usually applied without dilution to the area in whichtreatment is required. Typical carriers for granular formulationsinclude sand, fuller's earth, attapulgite clay, bentonite clays,montmorillonite clay, vermiculite, perlite, calcium carbonate, brick,pumice, pyrophyllite, kaolin, dolomite, plaster, wood flour, ground corncobs, ground peanut hulls, sugars, sodium chloride, sodium sulphate,sodium silicate, sodium borate, magnesia, mica, iron oxide, zinc oxide,titanium oxide, antimony oxide, cryolite, gypsum, diatomaceous earth,calcium sulphate and other organic or inorganic materials which absorbor which can be coated with the active compound. Granular formulationsnormally contain about 5% to about 25% active ingredients which mayinclude surface-active agents such as heavy aromatic naphthas, keroseneand other petroleum fractions, or vegetable oils; and/or stickers suchas dextrins, glue or synthetic resins.

Dusts are free-flowing admixtures of the active ingredient with finelydivided solids such as talc, clays, flours and other organic andinorganic solids which act as dispersants and carriers.

Microcapsules are typically droplets or granules of the activeingredient enclosed in an inert porous shell which allows escape of theenclosed material to the surroundings at controlled rates. Encapsulateddroplets are typically about 1 to 50 microns in diameter. The enclosedliquid typically constitutes about 50 to 95% of the weight of thecapsule and may include solvent in addition to the active compound.Encapsulated granules are generally porous granules with porousmembranes sealing the granule pore openings, retaining the activespecies in liquid form inside the granule pores. Granules typicallyrange from 1 mm to 1 cm and preferably 1 to 2 mm in diameter. Granulesare formed by extrusion, agglomeration or prilling, or are naturallyoccurring. Examples of such materials are vermiculite, sintered clay,kaolin, attapulgite clay, sawdust and granular carbon. Shell or membranematerials include natural and synthetic rubbers, cellulosic materials,styrene-butadiene copolymers, polyacrylonitriles, polyacrylates,polyesters, polyamides, polyureas, polyurethanes, and starch xanthates.

Other useful formulations for agrochemical applications include simplesolutions of the active ingredient in a solvent in which it iscompletely soluble at the desired concentration, such as acetone,alkylated naphthalenes, xylene and other organic solvents. Pressurizedsprayers, wherein the active ingredient is dispersed in finely-dividedform as a result of vaporization of a low boiling dispersant solventcarrier, may also be used.

In addition, further, other biocidally active ingredients orcompositions may be combined with the disclosed compound and used in themethods of the present disclosure and applied simultaneously orsequentially with the disclosed compound. When applied simultaneously,these further active ingredients may be formulated together with thedisclosed compound or mixed in, for example, the spray tank. Thesefurther biocidally active ingredients may be fungicides, herbicides,insecticides, bactericides, acaricides, nematicides and/or plant growthregulators.

Accordingly, the present disclosure provides for the use of acomposition in the methods of the present disclosure, said compositioncomprising (i) a disclosed compound and (i) a fungicide, (ii) anherbicide, (iii) an insecticide, (iv) a bactericide, (v) an acaricide,(vi) a nematicide and/or (vii) a plant growth regulator.

The herbicidal compositions of the present disclosure optionally canfurther comprise at least one non-auxin herbicide. The term “non-auxinherbicide” refers to an herbicide having a primary mode of action otherthan as an auxin herbicide. Representative examples of non-auxinherbicides include acetyl CoA carboxylase (ACCase) inhibitors,acetolactate synthase (ALS) inhibitors, acetohydroxy acid synthase(AHAS) inhibitors, photosystem II inhibitors, photosystem I inhibitors,protoporphyrinogen oxidase (PPO or Protox) inhibitors, carotenoidbiosynthesis inhibitors, enolpyruvyl shikimate-3-phosphate (EPSP)synthase inhibitor, glutamine synthetase inhibitor, dihydropteroatesynthetase inhibitor, mitosis inhibitors, and nucleic acid inhibitors;salts and esters thereof; racemic mixtures and resolved isomers thereof;and combinations thereof.

Representative examples of ACCase inhibitors include clethodim,clodinafop, fenoxaprop-P, fluazifop-P, quizalofop-P, and sethoxydim.

Representative examples of ALS or AHAS inhibitors include flumetsulam,imazamethabenz-m, imazamox, imazapic, imazapyr, imazaquin, imazethapyr,metsulfuron, prosulfuron, and sulfosulfuron.

Representative examples of photosystem I inhibitors include diquat andparaquat.

Representative examples of photosystem II inhibitors include atrazine,cyanazine, diuron, and metibuzin.

Representative examples of PPO inhibitors include acifluorofen,butafenacil, carfentrazone-ethyl, flufenpyr-ethyl, fluthiacet,flumiclorac, flumioxazin, fomesafen, lactofen, oxadiazon, oxyfluorofen,and sulfentrazone.

Representative examples of carotenoid biosynthesis inhibitors includeaclonifen, amitrole, diflufenican, mesotrione, and sulcotrione.

A representative example of an EPSP inhibitor is N-phosphonomethylglycine (glyphosate).

A representative example of a glutamine synthetase inhibitor isglufosinate.

A representative example of a dihydropteroate synthetase inhibitor isasulam.

Representative examples of mitosis inhibitors include acetochlor,alachlor, dithiopyr, S-metolachlor, and thiazopyr.

Representative examples of nucleic acid inhibitors include difenzoquat,fosamine, metham, and pelargonic acid.

In one aspect, the herbicidal compositions of the present disclosurefurther comprise a non-auxin herbicide selected from the groupconsisting of acetochlor, glyphosate, glufosinate, flumioxazin,fomesafen, and agriculturally acceptable salts thereof.

In one aspect, the herbicidal compositions of the present disclosurefurther comprise glyphosate, or an agriculturally acceptable saltthereof. Suitable glyphosate salts include, for example, the ammonium,diammonium, dimethylammonium, monoethanolamine, isopropylamine, andpotassium salts, and combinations thereof. In one aspect, the glyphosatesalts are selected from the group consisting of monoethanolamine,isopropylamine, and potassium salts, and combinations thereof.

In one aspect, the herbicidal compositions of the present disclosurefurther comprise glufosinate, or an agriculturally acceptable saltthereof.

In one aspect, the herbicidal compositions of the present disclosure canfurther comprise dicamba, or an agriculturally acceptable salt or esterthereof, and glyphosate, or an agriculturally acceptable salt thereof.In another aspect, the herbicidal compositions of the present disclosurecomprise dicamba, or an agriculturally acceptable salt thereof;glyphosate, or an agriculturally acceptable salt thereof; and anon-ammoniated, agriculturally acceptable acetate salt. Commerciallyavailable sources of glyphosate, and its agriculturally acceptablesalts, include those products sold under the trade names DURANGO® DMA®,HONCHO PLUS®, ROUNDUP POWERMAX®, ROUNDUP WEATHERMAX®, TRAXION®, andTOUCHDOWN®.

In one aspect, the herbicidal compositions of the present disclosure canfurther comprise 2,4-D, or an agriculturally acceptable salt or esterthereof, and glyphosate, or an agriculturally acceptable salt thereof.In another aspect, the herbicidal compositions of the present disclosurecomprise 2,4-D, or an agriculturally acceptable salt or ester thereof;glyphosate, or an agriculturally acceptable salt thereof; and anon-ammoniated, agriculturally acceptable acetate salt.

In some aspects, the disclosed herbicidal compositions can furthercomprise an additive such as a pesticide. Exemplary pesticides include,but are not limited to, 2,4-D, acetochlor, aclonifen, amicarbazone,4-aminopicolinic acid based herbicides, such as halauxifen,halauxifen-methyl, and those described in U.S. Pat. Nos. 7,314,849 and7,432,227 to Balko, et al., amidosulfuron, aminocyclopyrachlor,aminopyralid, aminotriazole, ammonium thiocyanate, anilofos, asulam,azimsulfuron, atrazine, beflubutamid, benazolin, benfuresate,bensulfuron-methyl, bentazon-sodium, benzofenap, bifenox,bispyribac-sodium, bromobutide, bromacil, bromoxynil, butachlor,butafenacil, butralin, butroxydim, carbetamide, cafenstrole,carfentrazone, carfentrazone-ethyl, chlormequat, clopyralid,chlorsulfuron, chlortoluron, cinidon-ethyl, clethodim,clodinafop-propargyl, clomeprop, clomazone, cloransulam-methyl,cyanazine, cyclosulfamuron, cycloxydim, cyhalofop-butyl, daimuron,dicamba, dichlobenil, dichlorprop-P, diclofop-methyl, diclosulam,diflufenican, diflufenzopyr, dimefuron, dimethachlor, diquat, diuron,S-ethyl dipropylcarbamothioate (EPTC), esprocarb, ethoxysulfuron,etobenzanid, fenoxaprop, fenoxaprop-ethyl,fenoxaprop-ethyl+isoxadifen-ethyl, fenoxaprop-P-ethyl, fenoxasulfone,fenquinotrione, fentrazamide, flazasulfuron, florasulam, fluazifop,fluazifop-P-butyl, flucarbazone, flucarbazone-sodium, flucetosulfuron(LGC-42153), flufenacet, flumetsulam, flumioxazin, flupyrsulfuron,flurochloridone, fluroxypyr, fluroxypyr-meptyl, flurtamone, glufosinate,glufosinate-ammonium, glyphosate, halosulfuron-methyl, haloxyfop-methyl,haloxyfop-R-methyl, hexazinone, imazamethabenz, imazamox, imazapic,imazapyr, imazaquin, imazethapyr, imazosulfuron, indanofan, indaziflam,iodosulfuron, iodosulfuron-ethyl-sodium, iofensulfuron, ipfencarbazone,isoproturon, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB,mecoprop-P, mefenacet, mesosulfuron, mesosulfuron-ethyl sodium,mesotrione, metamifop, metazochlor, metazosulfuron, metosulam,metribuzin, metsulfuron, metsulfuron-methyl, molinate, MSMA,napropamide, napropamide-M, orfurazon, orthosulfamuron, oryzalin,oxadiargyl, oxadiazon, oxazichlomefone, oxyfluorfen, paraquat,pendimethalin, penoxsulam, pentoxazone, pethoxamid, picloram,picolinafen, pinoxaden, pretilachlor, primisulfuron, profluazol,profoxydim, propanil, propaquizafop, propyrisulfuron, propoxycarbazone,propyzamide, prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl,pyrasulfotole, pyrazosulfuron-ethyl, pyrazolynate, pyribenzoxim(LGC-40863), pyributicarb, pyridate, pyriftalid, pyrimisulfan,pyroxsulam, pyroxasulfone, quinclorac, quinmerac, quizalofop-ethyl-D,quizalofop-P-ethyl, quizalofop-P-tefuryl, rimsulfuron, sethoxydim,simazine, sulfentrazone, sulfometuron, sulfosate, sulfosulfuron,tebuthiuron, tefuryltrione, tepraloxidim, terbacil, terbuthylazine,terbutryn, thenylchlor, thiazopyr, thifensulfuron,thifensulfuron-methyl, thiobencarb, topramezone, tralkoxydim,triafamone, triasulfuron, tribenuron, tribenuron-methyl, triafamone,triclopyr, and trifluralin, and agriculturally acceptable salts, cholinesalts, esters and mixtures thereof. In certain aspects, the additionalpesticide includes benzofenap, cyhalofop, daimuron, pentoxazone,esprocarb, pyrazosulfuron, butachlor, pretilachlor, metazosulfuron,bensulfuron-methyl, imazosulfuron, azimsulfuron, bromobutide,benfuresate, mesotrione, oxazichlomefone, and agriculturally acceptablesalts or esters thereof, or combinations thereof. In certain aspects,the additional pesticide includes triclopyr choline salt.

Methods of Using

The agricultural compositions disclosed herein can be applied in anyknown technique for applying herbicides. Exemplary applicationtechniques include, but are not limited to, spraying, atomizing,dusting, spreading, or direct application into water (in-water). Themethod of application can vary depending on the intended purpose. Insome aspects, the method of application can be chosen to ensure thefinest possible distribution of the compositions disclosed herein.

The agricultural compositions disclosed herein can be appliedpre-emergence (before the emergence of undesirable vegetation) orpost-emergence (i.e., during and/or after emergence of the undesirablevegetation). The composition can be applied, for example, to thevegetation as an in-water application to an irrigated potato field.

When the agricultural compositions are used in crops, the compositionscan be applied after seeding and before or after the emergence of thecrop plants. In some aspects, the compositions disclosed herein showgood crop tolerance even when the crop has already emerged, and can beapplied during or after the emergence of the crop plants. In someaspects, when the compositions are used in crops, the compositions canbe applied before seeding of the crop plants.

In some aspects, the compositions disclosed herein are applied tovegetation or an area adjacent the vegetation, or applied to soil, orapplied to/into water, for example to/into an irrigation water sourcefor a crop, to prevent the emergence or growth of vegetation by spraying(e.g., foliar spraying). In some aspects, the spraying techniques use,for example, water as carrier and spray liquor rates of from 10 litersper hectare (L/ha) to 2000 L/ha (e.g., from 50 L/ha to 1000 L/ha, orfrom 100 to 500 L/ha). In some aspects, the compositions disclosedherein are applied by the low-volume or the ultra-low-volume method,wherein the application is in the form of micro granules. In someaspects, the compositions disclosed herein can be applied as dryformulations (e.g., granules, WDGs) into water.

The compositions and methods disclosed herein can be used to controlundesired vegetation in a variety of crop and non-crop applications. Insome aspects, the compositions and methods disclosed herein can be usedfor controlling undesired vegetation in potatoes (e.g., in potatoesgrown from seed potatoes, potato plants grown from callus or tissueculture, or the like).

Method for Scaling Production of Nitroreductase Enzymes

In one aspect, disclosed herein are methods for scaling production ofnitroreductase enzymes for commercial use and/or for use in furtherstudies. In a further aspect, the nitroreductases of interest can befrom any organism that produces a nitroreductase enzyme. In a furtheraspect, the nitroreductase can be from Haemophilus influenzae,Actinobacillus indolicus, Avibacterium paragallinarum, Mannheimiasucciniproducens, Staphylococcus arlettae, Actinobacillus succinogenes,Arcobacter molloscorum, or a related organism. In one aspect, thenitroreductase is from H. influenzae. In some aspects, thenitroreductase gene has SEQ ID NO: 1. In any of the above aspects,primers for amplifying the chosen nitroreductase gene and/or cDNA can bechosen by the skilled artisan. In one aspect, the primers can be SEQ IDNO: 2 (NfB-Ndel-F) and SEQ ID NO: 3 (NfB-HindIII-R) or other primersmatching the ends of the area of DNA to be copied using the polymerasechain reaction (PCR). PCR can be carried out using establishedprotocols. Following PCR, in some aspects, the PCR product can bepurified by known techniques and digested with restriction enzymes suchas, for example, Ndel and HindIII, where the restriction enzymes haverecognition sites in the primers.

In a further aspect, a plasmid can be selected for incorporation of thecloned gene. In one aspect, the plasmid is pWLneo, pSV2cat, pOG44, pXT1,pSG, pSVK3, pBSK, pBSKII, pUC, pUC19, pETDuet-1, or pET22b. In oneaspect, the plasmid is pET22b. In another aspect, the plasmid isdigested with the same restriction enzymes (e.g., Ndel and HindIII oranother pair). Following digestion, the amplified DNA and/or the plasmidcan be separated from unwanted side product, for example, by agarose gelelectrophoresis followed by purification and extraction techniques. Inanother aspect, digested cloned DNA (i.e., the nitroreductase gene) andthe digested plasmid can be ligated to form an expression vectorcontaining the DNA of interest. In one aspect, successful insertion ofthe cloned gene can be established by sequencing the expression vector.

In another aspect, established transformation protocols can be used inorder to insert the expression vector into a host bacterial cell suchas, for example, E. coli. In one aspect, successful transformation canbe assessed by culturing the host bacterial cells in a mediumincorporating an antibiotic such as, for example, ampicillin, where theexpression vector contains a gene for resistance to ampicillin oranother antibiotic and where only cells that have been transformed areresistant to the antibiotic. In a further aspect, followingtransformation, the host bacterial cells can be cultured in anyacceptable medium. In a further aspect, host bacterial cells arecultured in a medium containing an antibiotic so that non-transformedcells do not compete with transformed cells for resources. In any ofthese aspects, when a desired cell concentration is reached, proteinexpression can be induced. In one aspect, the desired cell concentrationis from about 0.4 to 0.8 at 600 nm (i.e., OD₆₀₀ is from about 0.4 toabout 0.8), or is about 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, orabout 0.8, or a combination of any of the foregoing values, or a rangeencompassing any of the foregoing values. In one aspect, OD₆₀₀ is about0.6. In another aspect, protein expression can be induced with isopropylβ-D-1-thiogalactopyranoside (IPTG). In one aspect, induction can occurat a temperature of from about 15 to about 20° C., or at about 15, 16,17, 18, 19, or about 20° C., or a combination of any of the foregoingvalues, or a range encompassing any of the foregoing values. In oneaspect, induction occurs at about 18° C. In another aspect, inductiontakes place with shaking at from about 150 to about 250 rpm, or about150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or about 250 rpm, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, induction takes place withshaking at 190 rpm. In still another aspect, induction occurs for fromabout 16 to about 24 hours, or for about 16, 17, 18, 19, 20, 21, 22, 23,or about 24 hours, or a combination of any of the foregoing values, or arange encompassing any of the foregoing values. In one aspect, inductionoccurs for about 20 hours.

In any of the above aspects, following induction of protein production,cells are centrifuged and pellets produced. In one aspect,centrifugation is conducted at from about 4000 to about 6000 rpm, orabout 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000,5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, or about 6000 rpm,or a combination of any of the foregoing values, or a range encompassingany of the foregoing values. In one aspect, centrifugation is conductedat about 5000 rpm. In another aspect, centrifugation is conducted forfrom about 5 to about 15 minutes, or for about 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or about 15 minutes, or a combination of any of theforegoing values, or a range encompassing any of the foregoing values.In one aspect, centrifugation is conducted for about 10 min. In stillanother aspect, centrifugation is conducted at a temperature of fromabout 2 to about 8° C., or at about 2, 3, 4, 5, 6, 7, or about 8° C., ora combination of any of the foregoing values, or a range encompassingany of the foregoing values. In one aspect, centrifugation is conductedat about 4° C. In any of the above aspects, following centrifugation,cell pellets can be resuspended in an appropriate lysis buffer. In oneaspect, the lysis buffer can include 25 mM Tris·HCl, 100 mM NaCl, 10 mMimidazole, 3 mM β-mercaptoethanol, and 10% glycerol, with a pH of about7.5. Slight variations and modifications of these values are alsoeffective. In another aspect, the cell biomass:buffer volume ratio canbe from about 1:1 to about 1:10, or can be about 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, or about 1:10, or a combination of any of theforegoing values or a range encompassing any of the foregoing values. Inone aspect, the cell biomass:buffer volume ratio is about 1:4. In astill further aspect, following contact with the lysis buffer, solubleproteins can be released from the cell pellets by sonication. In stillanother aspect, following sonication, centrifugation can again beperformed to collect the soluble proteins. In one aspect, centrifugationoccurs at from about 15,000 to about 20,000 rpm, or at about 15,000,15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,or about 20,000 rpm, or a combination of any of the foregoing values ora range encompassing any of the foregoing values. In one aspect,centrifugation occurs at about 18,000 rpm. In another aspect,centrifugation is conducted at a temperature of from about 2 to about 8°C., or at about 2, 3, 4, 5, 6, 7, or about 8° C., or a combination ofany of the foregoing values, or a range encompassing any of theforegoing values. In one aspect, centrifugation is conducted at about 4°C. In still another aspect, centrifugation can be carried out for fromabout 30 minutes to about 1 hour, or for about 30, 35, 40, 45, 50, 55,or about 60 minutes, or a combination of any of the foregoing values, ora range encompassing any of the foregoing values. In one aspect,centrifugation is conducted for about 40 min.

In another aspect, further purification can be accomplished using anappropriate resin. In one aspect, a Ni-NTA agarose resin can be used forprotein purification using procedures established in the art. In someaspects, the resin suppliers also provide instructions for using theresins. In one aspect, purified recombinant proteins can be exchangedinto a storage buffer following purification. In one aspect, the storagebuffer can include at least the following components: 25 mM Tris·HCl, 50mM NaCl, 10% glycerol. Variations of these concentrations are alsoenvisioned. In another aspect, the storage buffer has pH 8.0. In stillanother aspect, a desalting column such as, for example, a PD-10 column,can be used to exchange the recombinant proteins into the storagebuffer. In any of the above aspects, following purification and bufferexchange, the recombinant nitroreductase enzymes can be aliquoted andstored at −80° C. until use. In one aspect, protein concentrations canbe determined by any method known in the art including, but not limitedto, UV-Vis spectrophotometry. In some aspects, SDS-PAGE analysis oranother method can be used to show the recombinant protein has theexpected size.

Compositions Containing Nitroreductase Genes and/or Proteins

One or more of the polynucleotides encoding a nitroreductase gene and/orone or more nitroreductase proteins can be provided as an externalcomposition such as a spray or powder to the plant, plant part, seed, apest, or an area of cultivation. In another example, a plant istransformed with a DNA construct or expression cassette for expressionof at least one nitroreductase gene. In either composition, thenitroreductase gene, when contacted by a thaxtomin, can reduce the nitrogroup on the thaxtomin and render it non-phytotoxic. It is recognizedthat the composition can comprise a cell (such as plant cell or abacterial cell), in which a polynucleotide encoding the nitroreductasegene is stably incorporated into the genome and operably linked topromoters active in the cell. Compositions comprising a mixture ofcells, some cells expressing at least one nitroreductase gene are alsoencompassed. In other embodiments, compositions comprising thenitroreductase genes and/or proteins are not contained in a cell. Insuch embodiments, the composition can be applied to an area inhabited bya pathogenic bacterium such as one that secretes thaxtomins. In oneembodiment, the composition is applied externally to a plant (i.e., byspraying a field or area of cultivation) to protect the plant from thepathogenic bacterium. Methods of applying polynucleotides and/orproteins in such a manner are known to those of skill in the art.

The composition comprising the nitroreductase gene and/or protein can beformulated in an agriculturally suitable and/or environmentallyacceptable carrier. Such carriers can be any material that the animal,plant or environment to be treated can tolerate. Furthermore, thecarrier must be such that the composition remains effective atcontrolling a pathogenic microorganism and/or reducing plant damage fromthaxtomins secreted by the microorganism. Examples of such carriersinclude water, saline, Ringer's solution, dextrose or other sugarsolutions, Hank's solution, and other aqueous physiologically balancedsalt solutions, phosphate buffer, bicarbonate buffer and Tris buffer. Inaddition, the composition may include compounds that increase thehalf-life of a composition. Various insecticidal formulations can alsobe found in, for example, US Publications 2008/0275115, 2008/0242174,2008/0027143, 2005/0042245, and 2004/0127520, each of which is hereinincorporated by reference.

It is recognized that the polynucleotides comprising sequences encodingthe nitroreductase gene can be used to transform organisms to providefor host organism production of this components, and subsequentapplication of the host organism to the environment of the targetpathogenic microorganisms. Such host organisms include baculoviruses,bacteria, and the like. In this manner, the combination ofpolynucleotides encoding the nitroreductase gene may be introduced via asuitable vector into a microbial host, and said host applied to theenvironment, or to plants or animals.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may be stablyincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

Microbial hosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the sequences encoding the nitroreductase protein, and desirably,provide for improved protection of the components from environmentaldegradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacterxyli andAzotobacter vinlandir, and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

A number of ways are available for introducing the polynucleotidecomprising the nitroreductase gene into the microbial host underconditions that allow for stable maintenance and expression of suchnucleotide encoding sequences. For example, expression cassettes can beconstructed which include the nucleotide constructs of interest operablylinked with the transcriptional and translational regulatory signals forexpression of the nucleotide constructs, and a nucleotide sequencehomologous with a sequence in the host organism, whereby integrationwill occur, and/or a replication system that is functional in the host,whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include, but arenot limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2;Sambrook et al. (2000); Molecular Cloning: A Laboratory Manual (3rd ed.;Cold Spring Harbor Laboratory Press, Plainview, NY); Davis et al. (1980)Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, Cold SpringHarbor, NY); and the references cited therein.

Suitable host cells include the prokaryotes and the lower eukaryotes,such as fungi. Illustrative prokaryotes, both Gram-negative andGram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia,Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such asRhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia,Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae;Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceaeand Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetesand Ascomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of the present disclosure include ease of introducing thecoding sequence into the host, availability of expression systems,efficiency of expression, stability in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apathogen-control microcapsule include protective qualities, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorulaspp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp.,phylloplane organisms such as Pseudomonas spp., Erwinia spp., andFlavobacterium spp., and other such organisms, including Pseudomonasaeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillusthuringiensis, Escherichia coli, Bacillus subtilis, and the like.

The sequences encoding the nitroreductases encompassed by the presentdisclosure can be introduced into microorganisms that multiply on plants(epiphytes) to deliver these components to potential target pests.Epiphytes, for example, can be gram-positive or gram-negative bacteria.

The nitroreductase gene can be fermented in a bacterial host and theresulting bacteria processed and used as a microbial spray in the samemanner that Bacillus thuringiensis strains have been used asinsecticidal sprays. Any suitable microorganism can be used for thispurpose. By way of example, Pseudomonas has been used to expressBacillus thuringiensis endotoxins as encapsulated proteins and theresulting cells processed and sprayed as an insecticide Gaertner et al.(1993), in Advanced Engineered Pesticides, ed. L. Kim (Marcel Decker,Inc.).

Alternatively, the components of the present disclosure are produced byintroducing heterologous genes into a cellular host. Expression of theheterologous sequences results, directly or indirectly, in theintracellular production of the nitroreductase protein(s). Thesecompositions may then be formulated in accordance with conventionaltechniques for application to the environment hosting a target pest,e.g., soil, water, and foliage of plants. See, for example, EPA 0192319,and the references cited therein.

As disclosed herein, a transformed microorganism can be formulated withan acceptable carrier into separate or combined compositions that are,for example, a suspension, a solution, an emulsion, a dusting powder, adispersible granule, a wettable powder, and an emulsifiable concentrate,an aerosol, an impregnated granule, an adjuvant, a coatable paste, andalso encapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients disclosed herein (i.e., at least one nitroreductase enzyme)are normally applied in the form of compositions and can be applied tothe crop area, plant, or seed to be treated. For example, thecompositions may be applied to grain in preparation for or duringstorage in a grain bin or silo, etc. The compositions may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient or a composition that contains at leastone nitroreductase enzymeinclude, but are not limited to, foliarapplication, seed coating, and soil application. The number ofapplications and the rate of application depend on the intensity ofinfestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono- or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions comprising the nitroreductase proteins can be in asuitable form for direct application or as a concentrate of primarycomposition that requires dilution with a suitable quantity of water orother dilutant before application.

The compositions (including the transformed microorganisms) can beapplied to the environment of a plant pathogenic microorganism (e.g., aStreptomyces species that produces a thaxtomin) by, for example,spraying, atomizing, dusting, scattering, coating or pouring,introducing into or on the soil, introducing into irrigation water, byseed treatment or general application or dusting at the time when thepest has begun to appear or before the appearance of pests as aprotective measure. For example, the composition(s) and/or transformedmicroorganism(s) may be mixed with grain to protect the grain duringstorage. It is generally important to obtain good control of pathogenicmicroorganisms in the early stages of plant growth, as this is the timewhen the plant can be most severely damaged. In some aspects, thaxtominscan be particularly damaging to emergent plant seedlings. Thecompositions can conveniently contain another insecticide if this isthought necessary. In an embodiment of the present disclosure, thecomposition(s) is applied directly to the soil, at a time of planting,in granular form of a composition of a carrier and dead cells of aBacillus strain or transformed microorganism of the present disclosure.Another embodiment is a granular form of a composition comprising anagrochemical such as, for example, an herbicide, an insecticide, afertilizer, in an inert carrier, and dead cells of a Bacillus strain ortransformed microorganism of the present disclosure.

Now having described the aspects of the present disclosure, in general,the following Examples describe some additional aspects of the presentdisclosure. While aspects of the present disclosure are described inconnection with the following examples and the corresponding text andfigures, there is no intent to limit aspects of the present disclosureto this description. On the contrary, the intent is to cover allalternatives, modifications, and equivalents included within the spiritand scope of the present disclosure.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of thepresent disclosure and are not intended to limit the scope of what theinventors regard as their disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1: Materials and Methods Materials

Molecular biology reagents and enzymes were purchased from FisherScientific. Primers were ordered from Sigma-Aldrich. NfsB DNA fragmentwas synthesized by Eurofins. Thaxtomins were isolated according toprevious methods using the engineered S. albus strain. Other chemicalsand solvents were purchased from Sigma-Aldrich or Fisher Scientific. DNAsequencing was performed at Eurofins. A Shimadzu Prominence UHPLC system(Kyoto, Japan) fitted with an Agilent Poroshell 120 EC-C18 column (2.7μm, 4.6×50 mm) coupled with a PDA detector was used for HPLC analysis.For semi-preparative HPLC isolation of compound 5, an Agilent ZORBAXRX-C18 column (5 μm, 4.6×250 mm) was used. NMR spectra of compound 5were recorded in CD₃OD on a Bruker 600 MHz spectrometer at theUniversity of Florida, Gainesville, FL, USA.

HPLC Methods

The HPLC column (Agilent Poroshell 120 EC-C18, 2.7 μm, 4.6×50 mm) waskept at 30° C. was eluted first with 10% solvent B (acetonitrile with0.1% formic acid) for 2 min and then with a linear gradient of 10-50%solvent B in 8 min, followed by another linear gradient of 50-99%solvent B in 5 min. After eluting in 99% solvent B for 3 min, the linergradient of 99-10% solvent B in 1 min was used. The column was furtherre-equilibrated with 10% solvent B for 1 min. The flow rate was set as0.5 mL/min, and the products were detected at 380 nm or 280 nm with aPDA detector. For semi-preparative HPLC isolation of compound 5, thecolumn (Agilent ZORBAX RX-C18, 5 μm, 4.6×250 mm) kept at 30° C. waseluted first with 25% solvent B (acetonitrile) for 9.5 min and then witha linear gradient of 25-99% solvent B for 1 min, followed by cleaningwith 99% solvent B for 2.5 min. The column was then eluted with a lineargradient of 99-25% solvent B for 0.5 min and re-equilibrated with 25%solvent B for 0.5 min. The flow rate was set at 1 mL/min and theproducts were detected at 280 nm with a PDA detector. All isolates werecombined, concentrated, freeze-dried, and then weighed.

LC-MS Analysis

A SHIMADZU Prominence UPLC system fitted with an Agilent Poroshell 120EC-C18 column (2.7 μm, 4.6×50 mm) coupled with a Linear Ion TrapQuadrupole LC/MS/MS Mass Spectrometer system was used in the studies.The column was eluted with 10% solvent B (acetonitrile with 0.1% formicacid) for 2 min and then with a linear gradient of 10-50% solvent B in 8min, followed by another linear gradient of 50-99% solvent B in 5 min.After eluting in 99% solvent B for 3 min, the liner gradient of 99-10%solvent B in 1 min was used. The column was further re-equilibrated with10% solvent B for 1 min. The flow rate was set as 0.5 mL/min. For MSdetection, turbo spray conditions were identical for all chemicals(curtain gas: 30 psi; ion spray voltage: 5500 V; temperature: 600° C.;ion source gas 1: 50 psi; ion source gas 2: 60 psi).

Radish Seedling Assay

Serial concentrations of thaxtomins in DMSO were added into 20 mL of1.5% warm agar solution with gentle agitation to the finalconcentrations range from 0 to 2 μM. DMSO was included as a negativecontrol. The solution was then poured into the plate for solidificationat room temperature for 30 min. Radish seeds (Burpee) were surfacedisinfested, pregerminated, and selected when the radicle was 1±2 mm andjust emerged from the seed coat. Six radish seedlings were equallylocated on the surface of each plate with the root ends all pointed inthe same direction. Agar plates were covered and sealed with Parafilm.The seedlings in the agar plates grew at room temperature underfluorescent lighting (12 h per day for 7 days), and then the seedlingwith average size of all seedlings in the same plate was selected forcomparison.

Example 2: Preparation of Recombinant NfsB

The NfsB gene from Haemophilus influenzae (SEQ ID NO: 1) was synthesizedand then amplified via PCR reaction by using a pair of primers,NfB-Ndel-F (SEQ ID NO: 2) and NfB-Hindlll-R (SEQ ID NO: 3). The PCRproduct was purified and digested by the restriction enzymes Ndel andHindIII. The expression vector pET22b was also digested with Ndel andHindIII. Digested PCR product and plasmid were separated on agarose geland corresponding bands were purified for the ligation to generate theexpression construct pET22b-nfsB, which was sequenced to confirmsuccessful ligation. E. coli BL21 GOLD transformation, proteinexpression, and purification followed previously established protocols.Briefly, E. coli cells harboring the expression constructs were culturedin Terrific broth medium supplemented with ampicillin at 37° C., 250rpm. After OD₆₀₀ reached 0.6, protein expression was induced withisopropyl β-D-1-thiogalactopyranoside (IPTG; 0.1 mm) at 18° C., 190 rpmfor 20 h. Cell pellets were then collected after centrifugation (5000rpm, 10 min, and 4° C.). To purify recombinant proteins, cell pelletswere resuspended in lysis buffer (cell biomass/volume=1:4; 25 mMTris·HCl, pH 7.5, 100 mM NaCl, 10 mM imidazole, 3 mM β-mercaptoethanol,and 10% glycerol). Soluble proteins were released by sonication andcollected by centrifugation at 18000 rpm at 4° C. for 40 min. Ni-NTAagarose resin (Thermo) was then used for protein purification. Purifiedrecombinant proteins were exchanged into a storage buffer (25 mMTris·HCl, pH 8.0, 50 mM NaCl, and 10% glycerol) by using a PD-10 column,aliquoted, and stored at −80° C. until use. Protein concentrations weredetermined by UV/Vis spectrophotometry using a NanoDrop MicrovolumeSpectrophotometer (ThermoFisher Scientific). SDS-PAGE analysis indicatedthat the recombinant protein with a C-terminal Hiss tag showed theexpected size (26.7 kDa) (FIG. 2 ).

Example 3: Enzymatic Transformations of Thaxtomins into 4-AminoThaxtomins

Activity of recombinant NfsB on thaxtomins A-D (FIG. 1 ) was evaluated.The reaction mixture (100 μL) included 50 mM Tris-HCl, pH 8.0, 2 mMNADPH, 500 μM FMN, and 2 mM substrate. Reactions were initiated byadding 135 μM NfsB and further incubated at 25° C., 400 rpm for 5 hours.All four thaxtomin analogs were partially transformed into new productswhich have no UV absorbance at 380 nm and are more polar inreverse-phase HPLC analysis (FIG. 3A), an indicator of the absence ofthe nitro group. Further LC-MS analysis revealed that the molecularweights of the products were 30 Da smaller than corresponding substrates(FIG. 3B), and agreed with the calculated molecular weights of 4-aminocontaining compounds 5-8 (FIG. 1 ).

Enzymatic reaction conditions were optimized with thaxtomin A assubstrate. The enzyme showed the highest activity at 37° C. When coupledwith a glucose dehydrogenase (GDH)- or phosphite dehydrogenase(PTDH)-based NADPH regeneration system, the enzyme at 10 μM achievedgreater than 90% conversion of thaxtomin A (2 mM) at 37° C. in 8 hours.Furthermore, the clear cell lysates of E. coli BL21 strain expressingNfsB efficiently supported the nitro reduction reaction with thaxtomin Aas substrate. In addition, a crude extract of engineered thaxtominproducing strain Streptomyces aibus-thx2 was prepared; the extractcontained about 69% thaxtomin A based on HPLC analysis. Recombinant NfsBand its clear cell lysates both completely converted thaxtomin A (1) inthe crude extract into compound 5.

4-amino thaxtomin A (5): colorless solid; MS m/z 409.4 [M+H]+ (calcd.for C₂₂H₂₄N₄O₄, 409.2); ¹H NMR (600 MHz, CD₃OD) δ 7.14 (t, J=7.8 Hz,1H), 6.84 (t, J=7.8 Hz, 1H), 6.75-6.69 (m, 2H), 6.66-6.60 (m, 2H), 6.45(d, J=7.6 Hz, 1H), 6.32 (dd, J=7.4, 0.7 Hz, 1H), 4.38 (dd, J=9.2, 2.6Hz, 1H), 3.07 (d, J=13.5 Hz, 1H), 3.04-2.98 (m, 4H), 2.94 (d, J=13.5 Hz,1H), 2.53 (s, 3H), 1.45 (dd, J=15.4, 9.2 Hz, 1H). ¹³C NMR (150 MHz,CD₃OD):) δ 168.43, 168.41, 159.04, 141.80, 140.26, 137.12, 130.99,124.24, 123.92, 122.84, 118.73, 117.18, 115.80, 111.66, 106.36, 103.82,87.59, 65.47, 44.16, 35.24, 34.32, 28.78.

Example 4: Preparative Scale Synthesis of 4-Amino Thaxtomin A andStructure Characterization

To further elucidate the chemical structures of products in the NfsBreaction, the reaction with NfsB containing E. coli cell lysates and thecrude extract of thaxtomins as substrate was scaled up. In a 250 mLflask, the reaction mixture (100 mL) contained 7.5 mL of NfsB lysate(cell pellet from one liter TB culture generated 19 mL cell lysate), 2mL crude extract of thaxtomins (21.9 mg/mL in DMSO, 69% of thaxtomin A),5 mL of 50 mM Tris-HCl buffer at pH 8, 0.5 mL FMN (50 mM), 2 mL NADP+(50mM), 1 mL GDH (0.75 mM), 1.5 mL glucose (40%). The reaction wasincubated at 37° C. for 6 hours and then terminated by adding the samevolume of ethyl acetate. The products were extracted with ethyl acetatetwo additional times. The organic layers were combined, washed withwater, dried and then evaporated in vacuo. The resulting residue waspurified by HPLC and the fractions of targeted products were collectedand dried by lyophilization to afford 15.5 mg of solid compound 5. Theconversion yield was determined to be 55.6%. 1D and 2D NMR analysis ofthe isolated product revealed its structure as compound 5 (FIG. 1 ,FIGS. 4A-E, and Table 2).

TABLE 2 ¹H and ¹³C NMR data comparison of compounds 1 and 5

Thaxtomin A^(a) 5^(b) Position δ_(C), type δ_(H) (J in Hz) δ_(C), typeδ_(H) (J in Hz)  2 132.5, CH 6.95, s 124.2, CH , 6.64, s  3 110.5, C111.7, C  4 143.6, C 141.8, C  5 119.2, CH 7.84 (7.9, 1.0, dd) 106.4, CH6.32 (7.4, 0.7, dd)  6 121.0, CH 7.19 (8.0, t) 123.9, CH 6.84, (7.8, t) 7 118.6, CH 7.68 (8.1, 1.0, dd) 103.8, CH 6.73, m  8 119.8, C 117.2, C 9 141.1, C 140.3, C 10  33.5, CH₂ 1.62 (14.2, 8.9, dd);  34.3, CH₂ 1.45(15.4, 9.2, dd); 2.60 (14.1, 6.2,0.5, ddd) 3.01, m 11  64.6, CH 3.86(8.9, 6.3, dd)  65.5, CH 4.38 (9.2, 2.6, dd) 13 168.3, C 168.4, C 14 88.0, C  87.6, C 16 166.8, C 168.4, C 17  45.4, CH₂ 3.11 (13.4, d);3.32 (13.5, d)  44.2, CH₂ 2.94 (13.5, d); 3.07 (13.5, d) 18 137.4, C137.1, C 19 118.4, CH 6.71, m 118.7, CH 6.62, m 20 159.1, C 159.0, C 21115.8, CH 6.71, m 115.8, CH 6.73, m 22 131.2, CH 7.23 (8.1, t) 131.0, CH7.14 (7.8, t) 23 122.7, CH 6.71, m 122.8, CH 6.45 (7.6, t) N-12  28.5,CH₃ 3.03, s  28.8, CH₃ 3.02, s N-15  34.2, CH₃ 2.81, S  35.2, CH₃ 2.53,s ^(a)NMR data were reported in literature; ^(b)NMR spectra wererecorded in CDOD₃.

Example 5: Radish Seedling Assay of 4-Amino Thaxtomin A

A radish seedling assay was carried out to investigate the herbicidalactivity of serial concentrations of 4-amino thaxtomin A (compound 5).The assay also included DMSO as negative control and thaxtomin A (1) aspositive control. After seven days, the growth of radish seedlings onagar plate with compound 5 at both 0.05 μM and 2.0 μM was the same asthe negative control DMSO (FIG. 5 ). Expectedly, thaxtomin A (1)significantly inhibited the growth of radish seedlings at 2.0 μM. Theresults indicated that compound 5 has minimal to no herbicidal activityto radish seedlings. The diminished herbicidal activity is caused by thetransformation of the nitro group of thaxtomin A (1) into the aminegroup.

Example 6: Preparation of Recombinant NfsB R20A Mutant

The mutagenesis was done via overlapping PCR with four primers shown inTable 3. Briefly, the PCR reaction contained 2 μM of each primer, 0.1 mMof each dNTP (Thermo) and 0.5 ul Phusion high fidelity DNA polymerase(NEB) in 1X GC reaction buffer. Reaction conditions consisted of aninitial denaturation step at 98° C. for 30 s followed by 30 cycles of98° C. for 10 s, 61° C. for 30 s, and 72° C. for 30 s, and a finalextension of 72° C. for 5 min. The PCR product was separated on a 1%agarose gel, visualized by staining with SYBR safe and extracted using aGeneJET Gel Extraction Kit (Thermo). After gel purification and productconcentration measurement, the purified PCR products were used foroverlapping PCR. Equimolar amounts of purified fragments (around 100 ng)was added to a 25-uL PCR reaction. First, 15 PCR cycles were run withoutprimers. Second, 2 uM end primers (nfsbFNdel and nfsbRHindIII) wereadded to the reaction, which was then continued for 20 cycles. The PCRproduct was separated and purified on a 1% agarose gel. The purifiedproduct along with pET 22b was digested by Ndel and HindIII. The T4ligation reaction was performed with a product and plasmid molar ratioat 3:1; 4° C. overnight. An aliquot (2 ul) was then used to transform 50ul E. coli BL21-GOLD electro-competent cells and positive colonies wereselected on the LB agar medium with 100 ug/ml ampicillin. The mutationwas confirmed by sequencing the insert of the constructs isolated frompositive colonies. The recombinant NfsB R20A mutant was prepared from E.coli BL21-GOLD following the same procedure in the preparation of thewild type.

TABLE 3 Primers used to prepare an NsfB R20A  mutant Primer NameSequence (5′ to 3′) nfsbFNde1 ACTCATATGACTCAACTTACTCGTGAA (SEQ ID NO: 7) nfsbRHindIII ACTAAGCTTCCCCACCCATTTCACCACTTCA (SEQ ID NO: 8) nfsbR20A-F GCTCAACAGCGTATTACGACCC  (SEQ ID NO: 9)nfsbR20A-R GGGTCGTAATACGCTGTTGAGC  (SEQ ID NO: 10)

Example 7: R20 is Catalytically Important in Converting Thaxtomins

Based on sequence alignments with homologs, it was further proposed thatthe relatively conserved R20 of NfsB may be catalytically critical byinteracting with the nitro group of the substrate and potentially thecofactor FMN. A recombinant NfsB R20A mutant (FIG. 7A) was prepared inE. coli to test this hypothesis. The same concentrations of wild typeNfsB and its R20A mutant A were incubated with 1 mM thaxtomin for 5hours. LC-MS analysis revealed that the R20A mutant retained only about12.5% of the catalytic activity of the wild type (FIG. 7B), indicatingthe catalytic role of R20 residue. The potential role of the R20 in thebinding of cofactor FMN was further assessed. HPLC analysis identifiedthat the FMN content of R20A mutant was about 42% of the wild type.Together, these results demonstrated that the R20 residue of NfsB islikely involved in the binding of both substrate and cofactor. Furthermutagenesis of this and other residues potentially interacting with thesubstrate (e.g., A and B helices in FIG. 6 ) can likely develop NfsBmutants with improved activity and substrate specificity towardthaxtomins.

The present disclosure further includes the following aspects.

Aspect 1. A plant cell with stably integrated, recombinant DNAcomprising a nucleotide sequence that encodes a nitroreductase protein.

Aspect 2. The plant cell of aspect 1, wherein the plant cell furthercomprises a heterologous promoter that is functional in plant cells andthat is operably linked to the nucleotide sequence that encodes thenitroreductase protein.

Aspect 3. The plant cell of aspect 1 or 2, wherein the nucleotidesequence that encodes the nitroreductase protein is isolated fromHaemophilus influenzae, Actinobacillus indolicus, Avibacteriumparagallinarum, Mannheimia succiniproducens, Staphylococcus arlettae,Actinobacillus succinogenes, or Arcobacter molloscorum.

Aspect 4. The plant cell of aspect 3, wherein the nucleotide sequencethat encodes the nitroreductase protein is isolated from Haemophilusinfluenzae.

Aspect 5. The plant cell of any of aspects 1-4, wherein thenitroreductase protein is NfsB.

Aspect 6. The plant cell of any of aspects 1-5, wherein the nucleotidesequence that encodes the nitroreductase protein comprises at least 90%sequence identity with SEQ ID NO: 1.

Aspect 7. The plant cell of any of aspects 1-5, wherein the nucleotidesequence that encodes the nitroreductase protein comprises at least 95%sequence identity with SEQ ID NO: 1.

Aspect 8. The plant cell of any of aspects 1-5, wherein the nucleotidesequence that encodes the nitroreductase protein comprises at least 97%sequence identity with SEQ ID NO: 1.

Aspect 9. The plant cell of any of aspects 1-5, wherein thenitroreductase protein comprises SEQ ID NO: 4.

Aspect 10. The plant cell of aspect 9, wherein the nitroreductaseprotein further comprises a portion comprising at least 70% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 70%sequence identity with SEQ ID NO: 6.

Aspect 11. The plant cell of aspect 9, wherein the nitroreductaseprotein further comprises a portion comprising at least 75% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 75%sequence identity with SEQ ID NO: 6.

Aspect 12. The plant cell of aspect 9, wherein the nitroreductaseprotein further comprises a portion comprising at least 80% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 80%sequence identity with SEQ ID NO: 6.

Aspect 13. The plant cell of aspect 9, wherein the nitroreductaseprotein further comprises a portion comprising at least 85% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 85%sequence identity with SEQ ID NO: 6.

Aspect 14. A transgenic plant comprising a plurality of the plant cellof any of aspects 1-13.

Aspect 15. A transgenic seed comprising a plurality of the plant cell ofany of aspects 1-13.

Aspect 16. A transgenic plant callus comprising a plurality of the plantcell of any of aspects 1-13.

Aspect 17. A progeny plant grown from the seed of aspect 15 or thecallus of aspect 16.

Aspect 18. The progeny plant of aspect 17, wherein the progeny plantcomprises the nucleotide sequence that encodes a nitroreductase protein.

Aspect 19. The transgenic plant of aspect 14 or the progeny plant of 17,wherein the transgenic plant or progeny plant is a potato plant, a beetplant, a carrot plant, a parsnip plant, a radish plant, a rutabagaplant, a turnip plant, or a sweet potato plant.

Aspect 20. The transgenic plant or progeny plant of aspect 19, whereinthe plant is a potato plant.

Aspect 21. A vegetable harvested from the plant of aspect 19 or 20.

Aspect 22. A plant chromosomal DNA segment comprising a recombinantpolynucleotide flanked by native plant DNA, wherein the polynucleotideprovides for expression of at least a nitroreductase protein.

Aspect 23. A plant chromosomal DNA segment comprising a recombinant DNAconstruct for expressing a nitroreductase protein comprising contiguousamino acids comprising at least 90% sequence identity to SEQ ID NO: 4.

Aspect 24. The plant chromosomal DNA segment of aspect 23, wherein therecombinant DNA construct for expressing a nitroreductase proteincomprises contiguous amino acids comprising at least 95% sequenceidentity to SEQ ID NO: 4.

Aspect 25. The plant chromosomal DNA segment of aspect 23, wherein therecombinant DNA construct for expressing a nitroreductase proteincomprises contiguous amino acids comprising at least 97% sequenceidentity to SEQ ID NO: 4.

Aspect 26. The plant chromosomal DNA segment of any of aspects 23-25,wherein the recombinant DNA construct for expressing a nitroreductaseprotein comprises a segment of contiguous amino acids comprising atleast 70% sequence identity with SEQ ID NO: 5 and a segment ofcontiguous amino acids comprising at least 70% sequence identity withSEQ ID NO: 6.

Aspect 27. The plant chromosomal DNA segment of any of aspects 23-25,wherein the recombinant DNA construct for expressing a nitroreductaseprotein comprises a segment of contiguous amino acids comprising atleast 75% sequence identity with SEQ ID NO: 5 and a segment ofcontiguous amino acids comprising at least 75% sequence identity withSEQ ID NO: 6.

Aspect 28. The plant chromosomal DNA segment of any of aspects 23-25,wherein the recombinant DNA construct for expressing a nitroreductaseprotein comprises a segment of contiguous amino acids comprising atleast 80% sequence identity with SEQ ID NO: 5 and a segment ofcontiguous amino acids comprising at least 80% sequence identity withSEQ ID NO: 6.

Aspect 29. The plant chromosomal DNA segment of any of aspects 23-25,wherein the recombinant DNA construct for expressing a nitroreductaseprotein comprises a segment of contiguous amino acids comprising atleast 85% sequence identity with SEQ ID NO: 5 and a segment ofcontiguous amino acids comprising at least 85% sequence identity withSEQ ID NO: 6.

Aspect 30. A transgenic plant cell comprising the plant chromosomal DNAsegment of any of aspects 22-29.

Aspect 31. A method of improving resistance to at least one thaxtomin ina crop plant line comprising providing in the genome of the crop plantline the plant chromosomal DNA segment of any of aspects 22-29.

Aspect 32. The method of aspect 31, wherein the thaxtomin is secreted bya pathogenic microorganism.

Aspect 33. The method of aspect 32, wherein the pathogenic microorganismis Streptomyces scabies, Streptomyces turgidiscabies, Streptomycesacidiscabies, Streptomyces luridiscabiei, Streptomyces puniciscabiei,Streptomyces nieviscabei, Streptomyces ipomoea, or a combinationthereof.

Aspect 34. The method of aspect 33, wherein the pathogenic microorganismis Streptomyces scabies.

Aspect 35. The method of aspect 31, wherein the thaxtomin is exogenouslyapplied.

Aspect 36. The method of any of aspects 31-35, wherein the thaxtomin isthaxtomin A, thaxtomin B, thaxtomin C, thaxtomin D, or a combinationthereof.

Aspect 37. The method of aspect 36, wherein the thaxtomin is thaxtominA.

Aspect 38. A DNA construct comprising a nucleotide sequence encoding anitroreductase protein.

Aspect 39. The DNA construct of aspect 38, wherein the DNA constructfurther comprises a heterologous promoter that is functional in plantcells and that is operably linked to the nucleotide sequence thatencodes the nitroreductase protein.

Aspect 40. The DNA construct of aspect 38 or 39, wherein the nucleotidesequence that encodes the nitroreductase protein is isolated fromHaemophilus influenzae, Actinobacillus indolicus, Avibacteriumparagaffinarum, Mannheimia succiniproducens, Staphylococcus arlettae,Actinobacillus succinogenes, or Arcobacter molloscorum.

Aspect 41. The DNA construct of aspect 40, wherein the nucleotidesequence that encodes the nitroreductase protein is isolated fromHaemophilus influenzae.

Aspect 42. The DNA construct of any of aspects 38-41, wherein thenitroreductase protein is NfsB.

Aspect 43. The DNA construct of any of aspects 38-42, wherein thenucleotide sequence that encodes the nitroreductase protein comprises atleast 90% sequence identity with SEQ ID NO: 1.

Aspect 44. The DNA construct of any of aspects 38-42, wherein thenucleotide sequence that encodes the nitroreductase protein comprises atleast 95% sequence identity with SEQ ID NO: 1.

Aspect 45. The DNA construct of any of aspects 38-42, wherein thenucleotide sequence that encodes the nitroreductase protein comprises atleast 97% sequence identity with SEQ ID NO: 1.

Aspect 46. The DNA construct of any of aspects 38-42, wherein thenitroreductase protein comprises SEQ ID NO: 4.

Aspect 47. The DNA construct of aspect 46, wherein the nitroreductaseprotein further comprises a portion comprising at least 70% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 70%sequence identity with SEQ ID NO: 6.

Aspect 48. The DNA construct of aspect 46, wherein the nitroreductaseprotein further comprises a portion comprising at least 75% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 75%sequence identity with SEQ ID NO: 6.

Aspect 49. The DNA construct of aspect 46, wherein the nitroreductaseprotein further comprises a portion comprising at least 80% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 80%sequence identity with SEQ ID NO: 6.

Aspect 50. The DNA construct of aspect 46, wherein the nitroreductaseprotein further comprises a portion comprising at least 85% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 85%sequence identity with SEQ ID NO: 6.

Aspect 51. An expression cassette comprising the DNA construct of any ofaspects 38-50.

Aspect 52. The expression cassette of aspect 51, wherein the nucleotidesequence is operably linked to a heterologous promoter.

Aspect 53. A host cell comprising the DNA construct of any of aspects38-45 or the expression cassette of aspect 51 or 52.

Aspect 54. The host cell of aspect 53, wherein the host cell is abacterial cell.

Aspect 55. The host cell of aspect 53 or 54, wherein the host cellcomprises the expression cassette of aspect 51 or 52.

Aspect 56. A plant cell having stably incorporated into its genome aheterologous polynucleotide comprising a nucleotide sequence encoding anitroreductase protein, wherein the heterologous polynucleotidecomprises a nucleotide sequence comprising at least 90% sequenceidentity to SEQ ID NO: 1 or a variant or fragment thereof; wherein thenucleotide sequence encoding the nitroreductase protein, whentranscribed and translated, produces a protein capable of reducing anitro group on a thaxtomin.

Aspect 57. The plant cell of aspect 56, wherein the heterologouspolynucleotide comprises a nucleotide sequence comprising at least 95%sequence identity to SEQ ID NO: 1 or a variant or fragment thereof;wherein the nucleotide sequence encoding the nitroreductase protein,when transcribed and translated, produces a protein capable of reducinga nitro group on a thaxtomin.

Aspect 58. The plant cell of aspect 56, wherein the heterologouspolynucleotide comprises a nucleotide sequence comprising at least 97%sequence identity to SEQ ID NO: 1 or a variant or fragment thereof;wherein the nucleotide sequence encoding the nitroreductase protein,when transcribed and translated, produces a protein capable of reducinga nitro group on a thaxtomin.

Aspect 59. The plant cell of any of aspects 56-58, wherein thenitroreductase protein comprises SEQ ID NO: 4.

Aspect 60. The plant cell of aspect 59, wherein the nitroreductaseprotein further comprises a portion comprising at least 70% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 70%sequence identity with SEQ ID NO: 6.

Aspect 61. The plant cell of aspect 59, wherein the nitroreductaseprotein further comprises a portion comprising at least 75% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 75%sequence identity with SEQ ID NO: 6.

Aspect 62. The plant cell of aspect 59, wherein the nitroreductaseprotein further comprises a portion comprising at least 80% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 80%sequence identity with SEQ ID NO: 6.

Aspect 63. The plant cell of aspect 59, wherein the nitroreductaseprotein further comprises a portion comprising at least 85% sequenceidentity with SEQ ID NO: 5 and a portion comprising at least 85%sequence identity with SEQ ID NO: 6.

Aspect 64. The plant cell of any of aspects 56-63, wherein the thaxtominis secreted by Streptomyces scabies, Streptomyces turgidiscabies,Streptomyces acidiscabies, Streptomyces luridiscabiei, Streptomycespuniciscabiei, Streptomyces nieviscabei, Streptomyces ipomoea, or acombination thereof.

Aspect 65. The plant cell of aspect 64, wherein the thaxtomin issecreted by Streptomyces scabies.

Aspect 66. The plant cell of any of aspects 56-63, wherein the thaxtominis exogenously applied as a component of an agricultural composition.

Aspect 67. The plant cell of aspect 66, wherein the agriculturalcomposition is an herbicide.

Aspect 68. The plant cell of any of aspects 56-63, wherein the plantcell comprises the expression cassette of aspect 51 or 52.

Aspect 69. The plant cell of any of aspects 56-63, wherein thenucleotide sequence encoding a nitroreductase protein is operably linkedto a heterologous promoter.

Aspect 70. The plant cell of any of aspects 56-69, wherein the plantcell is from a dicot.

Aspect 71. The plant cell of aspect 70, wherein the dicot is a potatoplant, a beet plant, a carrot plant, a parsnip plant, a radish plant, arutabaga plant, a turnip plant, or a sweet potato plant.

Aspect 72. A plant or plant part comprising the plant cell of any ofaspects 56-63.

Aspect 73. A transgenic seed from the plant of aspect 72.

Aspect 74. A method for reducing plant damage due to a plant pathogenicorganism comprising providing to a plant or soil before or afterintroduction of a seed, bulb, tuber, bud, stem, corm, plant part, or aplant, a composition comprising the DNA construct of any of aspects38-50, the expression cassette of aspect 51 or 52, or the host cell ofany of aspects 53-55.

Aspect 75. A method for reducing the damage caused by common scab ofpotato to the roots of a plant comprising providing to a plant or soilbefore or after introduction of a seed, bulb, tuber, bud, stem, corm,plant part, or a plant, a composition comprising the DNA construct ofany of aspects 38-50, the expression cassette of aspect 51 or 52, or thehost cell of any of aspects 53-55.

Aspect 76. A method for reducing the damage caused by a thaxtomin to theroots of a plant comprising providing to a plant or soil before or afterintroduction of a seed, bulb, tuber, bud, stem, corm, plant part, or aplant, a composition comprising the DNA construct of any of aspects38-50, the expression cassette of aspect 51 or 52, or the host cell ofany of aspects 53-55.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations setforth for a clear understanding of the principles of the presentdisclosure. Many variations and modifications may be made to theabove-described embodiment(s) without departing substantially from thespirit and principles of the present disclosure. All such modificationsand variations are intended to be included herein within the scope ofthis disclosure and protected by the following claims.

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1. A plant cell with stably integrated, recombinant DNA comprising anucleotide sequence that encodes a nitroreductase protein capable ofreducing a nitro group of at least one thaxtomin.
 2. The plant cell ofclaim 1, wherein the nucleotide sequence that encodes the nitroreductaseprotein is isolated from Haemophilus influenzae.
 3. The plant cell ofclaim 1, wherein the nitroreductase protein is NfsB.
 4. The plant cellof claim 1, wherein the nucleotide sequence that encodes thenitroreductase protein comprises at least 90% sequence identity with SEQID NO:
 1. 5. The plant cell of claim 1, wherein the nitroreductaseprotein comprises SEQ ID NO:
 4. 6. The plant cell of claim 1, whereinthe nitroreductase protein comprises a portion comprising at least 70%sequence identity with SEQ ID NO: 5 and a portion comprising at least70% sequence identity with SEQ ID NO:
 6. 7. A transgenic plantcomprising a plurality of the plant cell of claim
 1. 8. A transgenicseed comprising a plurality of the plant cell of claim
 1. 9. (canceled)10. (canceled)
 11. (canceled)
 12. (canceled)
 13. A method of improvingresistance to at least one thaxtomin in a crop plant line, the methodcomprising providing in the genome of the crop plant line a plantchromosomal DNA segment comprising a recombinant polynucleotide flankedby native plant DNA, wherein the recombinant polynucleotide encodes anitroreductase protein capable of reducing a nitro group of the at leastone thaxtomin.
 14. The method of claim 13, wherein the thaxtomin issecreted by a pathogenic microorganism.
 15. The method of claim 14,wherein the pathogenic microorganism is Streptomyces scabies,Streptomyces turqidiscabies, Streptomyces acidiscabies, Streptomycesluridiscabiei, Streptomyces puniciscabiei, Streptomyces nieviscabei,Streptomyces ipomoea, or a combination thereof.
 16. The method of claim13, wherein the thaxtomin is thaxtomin A, thaxtomin B, thaxtomin C,thaxtomin D, or a combination thereof.
 17. A DNA construct comprising: anucleotide sequence encoding a nitroreductase protein capable ofreducing a nitro group of at least one thaxtomin; and a heterologouspromoter that is functional in plant cells and that is operably linkedto the nucleotide sequence that encodes the nitroreductase protein. 18.(canceled)
 19. The DNA construct of claim 17, wherein the nitroreductaseprotein is NfsB, wherein the nucleotide sequence that encodes thenitroreductase protein comprises at least 90% sequence identity with SEQID NO:
 1. 20. The DNA construct of claim 17, wherein the nitroreductaseprotein comprises a portion comprising at least 70% sequence identitywith SEQ ID NO: 5 and a portion comprising at least 70% sequenceidentity with SEQ ID NO:
 6. 21. The method of claim 13, wherein thenitroreductase protein is isolated from Haemophilus influenzae.
 22. Themethod of claim 13, wherein the nitroreductase protein is NfsB.
 23. Themethod of claim 13, wherein the nucleotide sequence that encodes thenitroreductase protein comprises at least 90% sequence identity with SEQID NO:
 1. 24. The method of claim 13, wherein the nitroreductase proteincomprises SEQ ID NO:
 4. 25. The method of claim 13, wherein thenitroreductase protein further comprises a portion comprising at least70% sequence identity with SEQ ID NO: 5 and a portion comprising atleast 70% sequence identity with SEQ ID NO:
 6. 26. The method of claim13, wherein the thaxtomin is exogenously applied.